Activators of the unfolded protein response

ABSTRACT

A set of small molecules ERα biomodulators that kill therapy-resistant ERα positive breast, ovarian, and endometrial cancer cells. These small molecules have increased therapeutic potential because of an increased ability to kill therapy-resistant breast cancer cells compared to BHPI and other conventional therapies (endocrine therapies, tamoxifen and fulvestrant/ICI). The new compounds do not only inhibit proliferation of the cancer cells but actually kills them, which prevents reactivation of tumors years later.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/693,641, filed Jul. 3, 2018, whichis incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.RO1DK071909 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Approximately 70% of breast cancers are ERα positive. Endocrine(hormonal) therapies for these tumors includes aromatase inhibitors thatblock estrogen production, tamoxifen which competes with estrogens forbinding to ERα and fulvestrant/Faslodex/ICI 182,780, which both competeswith estrogens and promotes ERα degradation. Although effectiveinitially, resistance sometimes develops in primary tumors and is nearlyuniversal in the metastatic setting. Although resistance mechanisms arediverse, recent studies show that approximately 30% of these metastatictumors harbor ERα mutations, most commonly ERαD538G and ERαY537S. Thereis abundant evidence that these tumors exhibit estrogen-independentproliferation and are therefore resistant to aromatase inhibitors thatblock estrogen production. They are also largely resistant to tamoxifenand partially resistant to fulvestrant. Notably, patients whosemetastatic tumors contain the ERαD538G mutation have a 6-month shortersurvival time, and those with the ERαY537S mutation have a 12-monthshorter survival time, than patients whose metastatic tumors containnon-mutated wild-type ERα. Therefore, efforts to developchemotherapeutic agents targeting breast cancer cells containing thesemutations are extensive and widespread.

The pathology of ERα positive breast cancer is unusual. While 5-yearsurvival rates are impressive, the tumors often recur 10-20 years afterinitial diagnosis. This is thought to be due to reactivation ofproliferation of dormant breast cancer cells. Thus, it is especiallyimportant to actually kill the tumor cells, and not allow them to remaindormant and susceptible to reactivation. Current endocrine therapies arecytostatic, not cytotoxic, and therefore do not kill residual breastcancer cells. This allows the cells to lie dormant and reactivate at alater date. Therapeutic options for these recurrent tumors are poor andmost breast cancer deaths are in patients with ERα positive tumors.

Ovarian cancer usually presents at an advanced stage. Tumors often recurafter surgery. Although 30-70% of ovarian tumors are ERα positive, andERα expression is associated with a poor outcome, endocrine therapy isineffective and recurrent tumors are usually treated with platinum-basedchemotherapy and paclitaxel. Although initially responsive, afterseveral cycles of treatment, tumors recur as resistant ovarian cancer,with poor therapeutic options. More than half of ovarian cancer patientsdie within 5 years. In ovarian cancer, a very common mechanism forresistance to paclitaxel and other chemotherapy agents is multidrugresistance; energy dependent drug efflux caused by overexpression ofATP-dependent efflux pumps, especially Multidrug Resistance Protein 1(MDR1)/P-glycoprotein/ABCB1. Despite intensive efforts, effectivenon-toxic MDR1 inhibitors have remained elusive.

Although many cancers of the uterine endometrium are ERα positive,endocrine therapy works poorly.

Accordingly, an important therapeutic goal is therefore to identify newsmall molecules that are cytotoxic, not merely cytostatic, and todevelop corresponding therapeutic methods.

SUMMARY

This disclosure provides the benefits of small molecules with animproved ability to actually kill therapy resistant breast cancer cellsthat have greatly increased therapeutic potential. Specifically, toprevent recurrence, it is critical to destroy the entire population ofgrowing and dormant therapy resistant breast cancer cells. Compared toBHPI, and to the endocrine therapies tamoxifen and fulvestrant/ICI, theactive enantiomer (−)C-105 has greatly increased ability to kill breastcancer cells. The compound (−)C-105 therefore shows dramaticallyincreased therapeutic potential (FIG. 4A-4C).

This disclosure provides a compound of Formula I:

or a salt or solvate thereof;

wherein

-   -   R¹, R², R³ and R⁴ are each independently H, halo, —OR^(A),        —SR^(A), —N(R^(A))₂, alkyl, cycloalkyl, heterocyclyl, aryl, or        heteroaryl;    -   A¹, A², A³ and A⁴ are each independently H, halo, or alkyl;    -   G¹ is halo, —OR^(B), —SR^(B), —S(═O)₂R^(B), or alkyl;    -   G² is halo, —OR^(C), —SR^(C), —S(═O)₂R^(C), or alkyl;    -   X and Z are each independently O, S, or —NR^(D); and    -   R^(A), R^(B), R^(C) and R^(D) are each independently H or alkyl,        wherein, when present, —OR^(B) and —OR^(C) are not both —OH;

wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl isoptionally substituted with one or more substituents.

The above compound can bind to the alpha estrogen receptor (ERα) andkill or inhibit the growth of cancer cells by hyperactivation of theunfolded protein response (UPR) in the endoplasmic reticulum.

Additionally, this disclosure provides a method of treating a cancercomprising administering to an ERα positive cancer subject in needthereof a therapeutically effective amount of the compound above,thereby treating the cancer in the subject.

The invention provides novel compounds of Formulas I-IV, intermediatesfor the synthesis of compounds of Formulas I-IV, as well as methods ofpreparing compounds of Formulas I-IV. The invention also providescompounds of Formulas I-IV that are useful as intermediates for thesynthesis of other useful compounds. The invention provides for the useof compounds of Formulas I-IV for the manufacture of medicaments usefulfor the treatment of cancer in a mammal, such as a human.

The invention provides for the use of the compositions described hereinfor use in medical therapy. The medical therapy can be treating cancer,for example, breast cancer, ovarian cancer, uterine cancer, cervicalcarcinoma, endometrial cancer, lung cancer, pancreatic cancer, prostatecancer, or colon cancer. The invention also provides for the use of acomposition as described herein for the manufacture of a medicament totreat a disease in a mammal, for example, cancer in a human. Themedicament can include a pharmaceutically acceptable diluent, excipient,or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1. Dose response study of the effect of (±)-105 on ERα positive andERα negative human cancer cells. Alamar blue 24-hour death assay todetermine IC₅₀ of compound 105.

FIG. 2. Dose response study comparing the ability of BHPI and (±)-105 tokill ERα positive T47D cells. Compound (±)-105 potently induces breastcancer cell death.

FIG. 3. Comparison of the ability of (±)-105 to kill ERα positive andits inability to kill ERα negative cells.

FIG. 4. (−)105 Potently and Quantitatively Kills Cancer Cells. MCF7(ER+) cells, 24 hour incubation, read out via Alamar Blue Fluorescence,Dead Control: Raptinal, n=3 (A, B). (−)-105 is effective whenadministered orally (C). Orthoptic Mouse Xenograft breast cancers of TYScells, Visualization by bioluminescent imaging. Treatment: 40 mg/kgdaily (−)-105 for 3 days either by oral gavage or by intraperitonealinjection.

FIG. 5. Chiral separation of (±)-105 showing resolution of individualenantiomers.

FIG. 6. (±)-105 chiral HPLC trace showing resolved peaks.

FIG. 7. (−)-105 Chiral HPLC trace of isolated enantiomer.

FIG. 8. (+)-105 Chiral HPLC trace of the other isolated enantiomer.

FIG. 9. The (−) enantiomer of 105 is active and the (+) enantiomer isinactive in T47D cell line.

FIG. 10. (−)-105 induces dose-dependent death of MCF-7 cells; (+)-105 isinactive.

FIG. 11. Dose response study shows (−)-105 is superior to BHPI forkilling of T47D cells.

FIG. 12. Dose Response Study shows (−)-105 is superior to BHPI inkilling MCF-7 and TYS cells.

FIG. 13. Dose response study shows (−)-105 is superior to BHPI andcurrent endocrine therapies in killing TDG-Luc cells.

FIG. 14. Dose response studies to determine IC₅₀'s for killing of cancercells by (−)-105.

FIG. 15. In long-term experiments (−)-105, but not BHPI, kills 100% ofTYS-Luc cells.

FIG. 16. In Long-term experiments that simulate therapy, (−)-105, butnot BHPI, eradicates, lethal, therapy resistant breast cancer cells.

FIG. 17. Bioluminescent imaging (BLI) using luciferase shows neareradication of therapy-resistant TYS-luc breast tumors in mice.

FIG. 18. (−)-105 kills breast cancer cells resistant to killing by BHPI.

FIG. 19. (−)-105 potently activates the UPR as shown by induction ofspliced XBP-1 mRNA.

FIG. 20. (−)-105 is superior to BHPI in inhibiting protein synthesis intherapy-resistant Caov-3 ERα positive ovarian cancer cells.

FIG. 21. Blocking the efflux of calcium from the endoplasmic reticulumwith 2-APB inhibits (−)-105-induced cancer cell death.

FIG. 22. −105 reduces intracellular ATP levels; this reduction in ATPlevels is blocked by inactivating the endoplasmic reticulum SERCA pumpwith thapsigargin.

FIG. 23. Blocking the decline in ATP levels with thapsigargin inhibits−105 induced cell death.

FIG. 24. Comparison of the ability of test compounds and BHPI to killTDG cells.

FIG. 25. Comparison of the ability of test compounds and BHPI to killTYS cells.

FIG. 26. Comparison of the ability of test compounds 4, 6, 105 and BHPIto kill TYS cells.

FIG. 27. Comparison of the ability of test compounds and BHPI to killT47D cells.

FIG. 28. Neither the test compounds nor BHPI kill ERα negativenon-tumorigenic MCF-10A breast cells.

FIG. 29. Evaluation of the ability of test compounds and BHPI to killT47D cells.

FIG. 30. Dose-response studies comparing the ability of BHPI and testcompounds to inhibit proliferation of T47D, TYS and TDG cells.

FIG. 31. (−)105 is highly selective for ER+ cancer cells, including vsthe deadly ER+ mutants.

FIG. 32. (−)₁₀₅ has the same mechanism of action (MOA) as BHPI.

FIG. 33. 105, but not BHPI, kills therapy-resistant ovarian CaOV-3ovarian cancer cells. (±)-105; Cell death assay: Automated Trypan Blueexclusion. (24 hours post (±)-105)

FIG. 34. (−)105 nearly eradicates ER+ drug-resistant tumors. Orthotopicbreast cancer model, TYS-Luc cells, 40 mg/kg (−)-105 i.p., daily. 7dPost injection image is a much longer exposure at higher gain thanbefore injection image.

FIG. 35. Extra PPT slide (−)-105 is Superior to BHPI in Long-termExperiments Simulating Cancer Therapy. Cells: T47D-luciferase.

FIG. 36. Discovery of SERK-F6 (also known as, C(−)-105, (−)-105,(R)-105, (−)-1, or (R)-1), a compound that kills ERα-positive cancercells. a, Chemical structures of BHPI and racemic compound 1 (i.e.(±)-1), and their cytotoxic activity on T47D breast cancer cells after24-hour incubations with concentrations as indicated in nM. Cells werestained with annexin V-FITC and PI dyes prior to analysis via flowcytometry. Error bars represent S.E.M of 3 independent replicates. b,SERK-F6 is cytotoxic to ERα-positive cell line, MCF-7, but has minimaleffects against ERα-negative cell line, MDA-MB-231. Cells were incubatedwith SERK-F6 or positive control, Raptinal, for 24 hours and thenstained with annexin V-FITC and PI dyes and analyzed via flow cytometry.Error bars represent S.E.M of 3 independent replicates. c, IC₅₀ valuesof SERK-F6 after 24-hour incubation against a panel of cancer cell linesgrouped by their reported ERα status. Cell viability was measured byAlamar blue fluorescence and set relative to a vehicle control and aquantitative dead control treated with Raptinal. Error bars representS.E.M of 3 independent experiments. d, Crystal violet staining of T47Dcells after 24-hour incubations with compounds and concentrations asindicated. Image is representative of 2 independent experiments. e,f,Long-term cell culture experiment with T47D, TDG, and TYS cells. Cellswere incubated for 2 weeks with compounds (1 μM), followed by compoundwashout, and cells were cultured for 2 months. Cell number wasdetermined by MTS.

FIG. 37. SERK-F6 is the active species that leads to eradication ofERα-positive cells. a, Trypan-blue exclusion assay after 24 hours ofcompound incubation across a panel of ERα-positive cell lines. b,Long-term cell culture experiment with MCF-7, MDG, and MYS cellsincubated with 1 μM of BHPI or SERK-F6.

FIG. 38. SERK-F6 activates the anticipatory UPR. a, Induction of sp-XBP1mRNA in T47D cells as a result of compound treatment after indicatedtimes. BHPI and SERK-F6 were dosed at 1 μM. mRNA levels were quantifiedby RT-PCR. Error bars represent S.E.M of 6 biological replicates. b,Intracellular ATP depletion (need details here), also need T47D datahere c, SERK-F6 treatment (1 μM) leads to rapid protein synthesisinhibition in T47D cells.

FIG. 39. SERK-F6 eradicates wild-type ERα tumors via hyperactivation ofaUPR. a, MCF-7 orthotopic tumors were established in Nu/J ovariectomizedmice supplemented with a 60 day E2 pellet (0.36 mg) and grown to ˜400mm³ (28 days to establish), randomized, then treated with vehicle daily(n=3), vehicle weekly (n=3), fulv. weekly (5 mg/mouse, s.c., n=6),SERK-F6 daily (10 or 40 mg/kg p.o.). Vehicle arms were averaged. pvalues: *: p≤0.05, **: p≤0.01, ***: p≤0.001, ****: p≤0.0001. Tumorimages were taken at the end of the study (day 21) and arerepresentative of all tumors (n=6). b-e, MCF-7 cells were orthotopicallybi-laterally grafted and tumors were grown until total tumor burden was˜400 mm³ prior to daily treatment with vehicle or SERK-F6 (40 mg/kgp.o.). Mice (n=5 per arm) were sacrificed after 3 days (b,c) and 7 days(d,e) and tumors harvested. Tumor burden is displayed as a sum of bothtumors (b,d). c,e, western blot analysis of aUPR markers, P-PERK andP-EIF2α, as well as ERα. Vinculin (Vin) was used as a loading control.Western blots displayed are representative of 4 technical replicates. E2pellets were present during the progression of all experimentsdisplayed. Error bars represent S.E.M.

FIG. 40. SERK-F6 is tolerated, reaches biologically relevantconcentrations, and crosses the blood-brain barrier in vivo. a, Serumconcentrations of SERK-F6 after indicated doses, route ofadministration, and time. Concentration was determined via LC/MS/MSanalysis. The average 24-hour IC₅₀ of SERK-F6 is 42 nM. b, Calculatedpharmacokinetic parameters for SERK-F6. AUC: Area Under the Curve,C_(max): maximum concentration, t_(1/2): half-life. c,d, Mice (CD-1)were treated with the indicated doses and times, then sacrificed andtheir serum and brains collected. Concentrations were determined viaLC/MS/MS analysis. The average blood per mouse was approximated as 58.5mL/kg. f-j, SERK-F6 treatment of orthotopic MCF-7 tumor bearing mice. f,Percent tumor change with comparison of Day 0 and Day 21 tumormeasurements. Treatment arms: vehicle daily (n=3), vehicle weekly (n=3),fulv. weekly (5 mg/mouse, s.c., n=6), SERK-F6 daily (10 or 40 mg/kgp.o.). Vehicle arms were combined. g, Mass of tumors harvested frommice. i, observed murine weight during this MCF-7 study (n=6). i,j,Tumor measurements for MCF-7 bilateral orthotopic model (n=4-5).

FIG. 41. SERK-F6 eradicates mutant ERα primary and metastatic breastcancer. a-c, TYS-luc. orthotopic tumors were established in NSGovariectomized mice (no E2 pellet, 60 days to establish) and dosed dailyas indicated (n=5-6). b, representative examples of bioluminescenceimages of SERK-F6 treated mice (7 days). d,e, TDG-luc. orthotopic tumorswere established in NSG ovariectomized mice (no E2 pellet, 120 days toestablish) and dosed daily as indicated (n=4-5). e, Bioluminescenceimage of SERK-F6 treated mouse with TDG-luc. tumor burden after 7 days.f, MYS-luc. orthotopic tumors in NSG ovariectomized mice (no E2 pellet,45 days to establish) and dosed daily as indicated (n=4-5). g, MDG-luc.orthotopic tumors in NSG ovariectomized mice (no E2 pellet, 45 days toestablish) and dosed daily as indicated (n=4-5). h, Metastatic modelwith MDG-luc. cells inject into the tail-vein of NSG ovariectomized mice(no E2 pellet, 1.5 months to establish) and mice were dosed daily asindicated (n=4-5).

FIG. 42. The results show SERK-F6-mediated eradication of mutant ERα isdependent on quantitative cell killing. a, Treatment was stopped after21 daily doses of SERK-F6 (40 mg/kg i.p.) and mice tumor burden wastracked via bioluminescence. b,c TYS tumor bearing mice weight duringSERK-F6 treatment. d, Mice with TYS tumors treated at a lower dose ofSERK-F6 (10 mg/kg p.o.) regrow tumors, however retreatment of SERK-F6 ata higher dose (40 mg/kg p.o.) leads to complete regression. e, miceweights observed during the TDG model. f, TDG-bearing mice were treatedfor 14 continuous days, and treatment was halted, allowing forobservation of tumor regrowth via bioluminescent imaging. g, TDG-luc.,orthotopic tumors were establish in NSG ovariectomized mice (no E2pellet, ˜120 days to establish) and dosed daily as indicated (n=5-6). h,Mice with TDG-luc. tumors treated at a lower dose of SERK-F6 (10 mg/kgp.o.) regrow tumors after 2 months of no treatment, however retreatmentof SERK-F6 at a higher dose (40 mg/kg i.p.) leads to completeregression. Error bars displayed are S.E.M.

FIG. 43. SERK-F6-mediated eradication of MYS/MDG tumors is dependent onquantitative cell killing. a-b, MYS-luc. orthotopic tumors wereestablish in NSG ovariectomized mice (no E2 pellet, ˜45 days toestablish) and dosed daily as indicated (n=5-6). b, Treatment ofMYS-luc. tumors were stopped after 21 daily doses of SERK-F6 and micetumor burden was tracked via bioluminescence. At day 60, regrown tumorswere dosed with SERK-F6 (40 mg/kg daily i.p.). c, representativebioluminescence images. c-d, MDG-luc. orthotopic tumors were establishin NSG ovariectomized mice (no E2 pellet, ˜45 days to establish) anddosed daily as indicated (n=5-6). Tumor volume was tracked via totalphoton flux. d, MDG-luc. tumors treated with 40 mg/kg SERK-F6 p.o. dailyregrew after treatment was stopped. Regrown tumors were retreated with40 mg/kg SERK-F6 i.p. and tumor regression was observed.

DETAILED DESCRIPTION

Cancer cells can remain quiescent for extended periods of time and thenreactivate. It is therefore desirable to kill the tumor cells, notmerely to prevent them from proliferating. This disclosure also providesthe description of various assays for testing compounds for the abilityto kill cancer cells, such as breast cancer cells.

This disclosure also identifies new compounds that are more effectivethan BHPI in killing breast cancer cells expressing both wild typeestrogen receptor a (ERα) and ERα mutations that are common inmetastatic breast cancer. These mutations are associated with resistanceto current therapies. Also, this disclosure identifies compounds activeagainst ovarian cancer cells, uterine cancer cells and other cancercells that contain ERα.

The discovery of compound C(−)-105 is related in structure to BHPI. Theabsolute configuration of the levorotatory enantiomer of discoveredcompound 105 was determined to have the (R)-configuration (i.e.,(R)-105). It was identified to be cytotoxic to cancer cells, but not bymerely inhibiting cancer cell growth. The phenotype of (R)-105 killscancer cells and in turn tumors. This finding is surprising in view ofthe compounds BHPI, fulvestrant, tamoxifen, and 01-15 because they donot kill cancer cells. These compounds merely slow cancer cell growth,i.e., they are cytostatic. Thus, (R)-105 was identified by its distinctcytotoxicity profile and ability to quantitatively kill cancer cellsin-vitro. Data collected from additional in-vivo studies have proven thecompound's effectiveness in the regression of tumors.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X. It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with any element described herein, and/or the recitation ofclaim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit. For example, one or more substituents on a phenylring refers to one to five substituents on the ring.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements. Whenvalues are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value without themodifier “about” also forms a further aspect.

The terms “about” and “approximately” are used interchangeably. Bothterms can refer to a variation of ±5%, ±10%, ±20%, or ±25% of the valuespecified. For example, “about 50” percent can in some embodiments carrya variation from 45 to 55 percent, or as otherwise defined by aparticular claim. For integer ranges, the term “about” can include oneor two integers greater than and/or less than a recited integer at eachend of the range. Unless indicated otherwise herein, the terms “about”and “approximately” are intended to include values, e.g., weightpercentages, proximate to the recited range that are equivalent in termsof the functionality of the individual ingredient, composition, orembodiment. The terms “about” and “approximately” can also modify theendpoints of a recited range as discussed above in this paragraph.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. It is thereforeunderstood that each unit between two particular units are alsodisclosed. For example, if 10 to 15 is disclosed, then 11, 12, 13, and14 are also disclosed, individually, and as part of a range. A recitedrange (e.g., weight percentages or carbon groups) includes each specificvalue, integer, decimal, or identity within the range. Any listed rangecan be easily recognized as sufficiently describing and enabling thesame range being broken down into at least equal halves, thirds,quarters, fifths, or tenths. As a non-limiting example, each rangediscussed herein can be readily broken down into a lower third, middlethird and upper third, etc. As will also be understood by one skilled inthe art, all language such as “up to”, “at least”, “greater than”, “lessthan”, “more than”, “or more”, and the like, include the number recitedand such terms refer to ranges that can be subsequently broken down intosub-ranges as discussed above. In the same manner, all ratios recitedherein also include all sub-ratios falling within the broader ratio.Accordingly, specific values recited for radicals, substituents, andranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for radicals andsubstituents. It will be further understood that the endpoints of eachof the ranges are significant both in relation to the other endpoint,and independently of the other endpoint.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, for use in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an effective amount can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a compound described herein, or anamount of a combination of compounds described herein, e.g., that iseffective to treat or prevent a disease or disorder, or to treat thesymptoms of the disease or disorder, in a host. Thus, an “effectiveamount” generally means an amount that provides the desired effect.

Alternatively, the terms “effective amount” or “therapeuticallyeffective amount,” as used herein, refer to a sufficient amount of anagent or a composition or combination of compositions being administeredwhich will relieve to some extent one or more of the symptoms of thedisease or condition being treated. The result can be reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga compound as disclosed herein required to provide a clinicallysignificant decrease in disease symptoms. An appropriate “effective”amount in any individual case may be determined using techniques, suchas a dose escalation study. The dose could be administered in one ormore administrations. However, the precise determination of what wouldbe considered an effective dose may be based on factors individual toeach patient, including, but not limited to, the patient's age, size,type or extent of disease, stage of the disease, route of administrationof the compositions, the type or extent of supplemental therapy used,ongoing disease process and type of treatment desired (e.g., aggressivevs. conventional treatment).

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” can extend to prophylaxis andcan include prevent, prevention, preventing, lowering, stopping orreversing the progression or severity of the condition or symptoms beingtreated. As such, the term “treatment” can include medical, therapeutic,and/or prophylactic administration, as appropriate.

As used herein, “subject” or “patient” means an individual havingsymptoms of, or at risk for, a disease or other malignancy. A patientmay be human or non-human and may include, for example, animal strainsor species used as “model systems” for research purposes, such a mousemodel as described herein. Likewise, patient may include either adultsor juveniles (e.g., children). Moreover, patient may mean any livingorganism, preferably a mammal (e.g., human or non-human) that maybenefit from the administration of compositions contemplated herein.Examples of mammals include, but are not limited to, any member of theMammalian class: humans, non-human primates such as chimpanzees, andother apes and monkey species; farm animals such as cattle, horses,sheep, goats, swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice and guineapigs, and the like. Examples of non-mammals include, but are not limitedto, birds, fish and the like. In one embodiment of the methods providedherein, the mammal is a human.

As used herein, the terms “providing”, “administering,” “introducing,”are used interchangeably herein and refer to the placement of thecompositions of the disclosure into a subject by a method or route whichresults in at least partial localization of the composition to a desiredsite. The compositions can be administered by any appropriate routewhich results in delivery to a desired location in the subject.

The compositions described herein may be administered with additionalcompositions to prolong stability and activity of the compositions, orin combination with other therapeutic drugs.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “substantially” as used herein, is a broad term and is used inits ordinary sense, including, without limitation, being largely but notnecessarily wholly that which is specified. For example, the term couldrefer to a numerical value that may not be 100% the full numericalvalue. The full numerical value may be less by about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 15%, or about 20%.

This disclosure provides methods of making the compounds andcompositions of the invention. The compounds and compositions can beprepared by any of the applicable techniques described herein,optionally in combination with standard techniques of organic synthesis.Many techniques such as etherification and esterification are well knownin the art. However, many of these techniques are elaborated inCompendium of Organic Synthetic Methods (John Wiley & Sons, New York),Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6; as well as standard organic reference texts suchas March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th Ed., by M. B. Smith and J. March (John Wiley & Sons, NewYork, 2001); Comprehensive Organic Synthesis. Selectivity, Strategy &Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry M. Trost,Editor-in-Chief (Pergamon Press, New York, 1993 printing); AdvancedOrganic Chemistry, Part B: Reactions and Synthesis, Second Edition, Caryand Sundberg (1983);

The formulas and compounds described herein can be modified usingprotecting groups. Suitable amino and carboxy protecting groups areknown to those skilled in the art (see for example, Protecting Groups inOrganic Synthesis, Second Edition, Greene, T. W., and Wutz, P. G. M.,John Wiley & Sons, New York, and references cited therein; Philip J.Kocienski; Protecting Groups (Georg Thieme Verlag Stuttgart, New York,1994), and references cited therein); and Comprehensive OrganicTransformations, Larock, R. C., Second Edition, John Wiley & Sons, NewYork (1999), and referenced cited therein.

As used herein, the term “substituted” or “substituent” is intended toindicate that one or more (for example, 1-20 in various embodiments,1-10 in other embodiments, 1, 2, 3, 4, or 5; in some embodiments 1, 2,or 3; and in other embodiments 1 or 2) hydrogens on the group indicatedin the expression using “substituted” (or “substituent”) is replacedwith a selection from the indicated group(s), or with a suitable groupknown to those of skill in the art, provided that the indicated atom'snormal valency is not exceeded, and that the substitution results in astable compound. Suitable indicated groups include, e.g., alkyl,alkenyl, alkynyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl,heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl,acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, andcyano. Additionally, non-limiting examples of substituents that can bebonded to a substituted carbon (or other) atom include F, Cl, Br, I,OR′, OC(O)N(R′)₂, CN, CF₃, OCF₃, R′, O, S, C(O), S(O), methylenedioxy,ethylenedioxy, N(R′)₂, SR′, SOR′, SO₂R′, SO₂N(R′)₂, SO₃R′, C(O)R′,C(O)C(O)R′, C(O)CH₂C(O)R′, C(S)R′, C(O)OR′, OC(O)R′, C(O)N(R′)₂,OC(O)N(R′)₂, C(S)N(R′)₂, (CH₂)₀₋₂NHC(O)R′, N(R′)N(R′)C(O)R′,N(R)N(R)C(O)OR′, N(R′)N(R′)CON(R′)₂, N(R′)SO₂R′, N(R)SO₂N(R)₂,N(R′)C(O)OR′, N(R′)C(O)R′, N(R′)C(S)R′, N(R′)C(O)N(R′)₂,N(R′)C(S)N(R′)₂, N(COR′)COR′, N(OR′)R′, C(═NH)N(R′)₂, C(O)N(OR′)R′, orC(═NOR′)R′ wherein R′ can be hydrogen or a carbon-based moiety, andwherein the carbon-based moiety can itself be further substituted. Whena substituent is monovalent, such as, for example, F or Cl, it is bondedto the atom it is substituting by a single bond. When a substituent ismore than monovalent, such as O, which is divalent, it can be bonded tothe atom it is substituting by more than one bond, i.e., a divalentsubstituent is bonded by a double bond; for example, a C substitutedwith O forms a carbonyl group, C═O, wherein the C and the O are doublebonded. Alternatively, a divalent substituent such as O, S, C(O), S(O),or S(O)₂ can be connected by two single bonds to two different carbonatoms. For example, O, a divalent substituent, can be bonded to each oftwo adjacent carbon atoms to provide an epoxide group, or the O can forma bridging ether group between adjacent or non-adjacent carbon atoms,for example bridging the 1,4-carbons of a cyclohexyl group to form a[2.2.1]-oxabicyclo system. Further, any substituent can be bonded to acarbon or other atom by a linker, such as (CH₂)_(n) or (CR′₂)_(n)wherein n is 1, 2, 3, or more, and each R′ is independently selected.

The term “halo” or “halide” refers to fluoro, chloro, bromo, or iodo.Similarly, the term “halogen” refers to fluorine, chlorine, bromine, andiodine.

The term “alkyl” refers to a branched or unbranched hydrocarbon having,for example, from 1-20 carbon atoms, and often 1-12, 1-10, 1-8, 1-6, or1-4 carbon atoms. As used herein, the term “alkyl” also encompasses a“cycloalkyl”, defined below. Examples include, but are not limited to,methyl, ethyl, 1-propyl, 2-propyl (iso-propyl), 1-butyl,2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl), 2-methyl-2-propyl(t-butyl), 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl,3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl,3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Thealkyl can be unsubstituted or substituted, for example, with asubstituent described below. The alkyl can also be optionally partiallyor fully unsaturated. As such, the recitation of an alkyl group caninclude both alkenyl and alkynyl groups. The alkyl can be a monovalenthydrocarbon radical, as described and exemplified above, or it can be adivalent hydrocarbon radical (i.e., an alkylene).

The term “cycloalkyl” refers to cyclic alkyl groups of, for example,from 3 to 10 carbon atoms having a single cyclic ring or multiplecondensed rings. Cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such as adamantyl,and the like. The cycloalkyl can be unsubstituted or substituted. Thecycloalkyl group can be monovalent or divalent and can be optionallysubstituted as described for alkyl groups. The cycloalkyl group canoptionally include one or more cites of unsaturation, for example, thecycloalkyl group can include one or more carbon-carbon double bonds,such as, for example, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,1-cyclohex-3-enyl, and the like.

The term “heterocycloalkyl” or “heterocyclyl” refers to a saturated orpartially saturated monocyclic, bicyclic, or polycyclic ring containingat least one heteroatom selected from nitrogen, sulfur, oxygen,preferably from 1 to 3 heteroatoms in at least one ring. Each ring ispreferably from 3 to 10 membered, more preferably 4 to 7 membered.Examples of suitable heterocycloalkyl substituents include pyrrolidyl,tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl,tetrahydropyranyl, morpholino, 1,3-diazapane, 1,4-diazapane,1,4-oxazepane, and 1,4-oxathiapane. The group may be a terminal group ora bridging group.

The term “aromatic” refers to either an aryl or heteroaryl group orsubstituent described herein. Additionally, an aromatic moiety may be abisaromatic moiety, a trisaromatic moiety, and so on. A bisaromaticmoiety has a single bond between two aromatic moieties such as, but notlimited to, biphenyl, or bipyridine. Similarly, a trisaromatic moietyhas a single bond between each aromatic moiety.

The term “aryl” refers to an aromatic hydrocarbon group derived from theremoval of at least one hydrogen atom from a single carbon atom of aparent aromatic ring system. The radical attachment site can be at asaturated or unsaturated carbon atom of the parent ring system. The arylgroup can have from 6 to 30 carbon atoms, for example, about 6-10 carbonatoms. In other embodiments, the aryl group can have 6 to 60 carbonsatoms, 6 to 120 carbon atoms, or 6 to 240 carbon atoms. The aryl groupcan have a single ring (e.g., phenyl) or multiple condensed (fused)rings, wherein at least one ring is aromatic (e.g., naphthyl,dihydrophenanthrenyl, fluorenyl, or anthryl). Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,naphthalene, anthracene, biphenyl, and the like. The aryl can beunsubstituted or optionally substituted.

The term “heteroaryl” refers to a monocyclic, bicyclic, or tricyclicring system containing one, two, or three aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Theheteroaryl can be unsubstituted or substituted, for example, with one ormore, and in particular one to three, substituents, as described in thedefinition of “substituted”. Typical heteroaryl groups contain 2-20carbon atoms in the ring skeleton in addition to the one or moreheteroatoms. Examples of heteroaryl groups include, but are not limitedto, 2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl,benzo[b]thienyl, benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl,cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl,imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl,isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl,oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl, quinoxalinyl,thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl, tetrazolyl,and xanthenyl. In one embodiment the term “heteroaryl” denotes amonocyclic aromatic ring containing five or six ring atoms containingcarbon and 1, 2, 3, or 4 heteroatoms independently selected fromnon-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H, O,alkyl, aryl, or (C₁-C₆)alkylaryl. In some embodiments, heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “enantiomerically enriched” (“ee”) as used herein refers tomixtures that have one enantiomer present to a greater extent thananother. Reactions that provide one enantiomer present to a greaterextent than another would therefore be “enantioselective” (ordemonstrate “enantioselectivity”). In one embodiment of the invention,the term “enantiomerically enriched” refers to a mixture having at leastabout 2% ee; in another embodiment of the invention, the term“enantiomerically enriched” refers to a mixture having at least about 5%ee; in another embodiment of the invention, the term “enantiomericallyenriched” refers to a mixture having at least about 20%; in anotherembodiment of the invention, the term “enantiomerically enriched” refersto a mixture having at least about 50%; in another embodiment of theinvention, the term “enantiomerically enriched” refers to a mixturehaving at least about 80%; in another embodiment of the invention, theterm “enantiomerically enriched” refers to a mixture having at leastabout 90%; in another embodiment of the invention, the term“enantiomerically enriched” refers to a mixture having at least about95%; in another embodiment of the invention, the term “enantiomericallyenriched” refers to a mixture having at least about 98%; in anotherembodiment of the invention, the term “enantiomerically enriched” refersto a mixture having at least about 99%. The term “enantiomericallyenriched” includes enantiomerically pure mixtures which are mixturesthat are substantially free of the species of the opposite opticalactivity or one enantiomer is present in very low quantities, forexample, 0.01%, 0.001% or 0.0001%.

The term “IC₅₀” is generally defined as the concentration required tokill 50% of the cells in 24 hours.

Throughout this disclosure the term “(−)-105”, which refers to thelevorotatory enantiomer of compound 105, may be used interchangeablywith terms C(−)-105, (−)-1, (R)-105, (R)-1, or SERK-F6. Similarly,(+)-105 is the dextrorotatory enantiomer, also known as (5)-105, (5)-1or (+)-1. Also, the term “(±)-105”, which refers to the racemate ofcompound 105, may be used interchangeably with the term (±)-1.

Embodiments of the Invention

This disclosure provides various embodiments of a compound of Formula I:

or a salt or solvate thereof; wherein

-   -   R¹, R², R³ and R⁴ are each independently H, halo, —OR^(A),        —SR^(A), —N(R^(A))₂, alkyl, cycloalkyl, heterocyclyl, aryl, or        heteroaryl;    -   A¹, A², A³ and A⁴ are each independently H, halo, or alkyl;    -   G¹ is halo, —OR^(B), —SR^(B), —S(═O)₂R^(B), or alkyl;    -   G² is halo, —OR^(C), —SR^(C), —S(═O)₂R^(C), or alkyl;    -   X and Z are each independently O, S, or —NR^(D); and    -   R^(A), R^(B), R^(C) and R^(D) are each independently H or alkyl,        wherein, when present, —OR^(B) and —OR^(C) are not both —OH;

wherein each alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl isoptionally substituted with one or more substituents.

In various embodiments of Formulas I-IV described herein, R⁵ is asubstituent in the position ortho to A¹. In various embodiments, R⁶ is asubstituent in the position ortho to A². In various embodiments, R⁷ is asubstituent in the position ortho to A³. In various embodiments, R⁸ is asubstituent in the position ortho to A⁴. In various embodiments, R⁵, R⁶,R⁷ and R⁸ are each independently H, halo, —OR^(A), —SR^(A), —N(R^(A))₂,alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In some embodiments, the compound is the (S)-enantiomer. In otherembodiments, the compound is the (R)-enantiomer. In additionalembodiments, R^(A), R^(B), R^(C) and R^(D) are each independently H or—(C₁-C₆)alkyl, and R², R³ and R⁴ are each independently H, halo, or—(C₁-C₆)alkyl. In some other embodiments, A¹, A², A³ and A⁴ are eachindependently H or halo, and G¹ is —OR^(B). In further embodiments, X is—NR^(D) and Z is O. In further embodiments, R^(A), R^(B), R^(C), R^(D),R¹, R², R³ and R⁴ are each independently —(C₂-C₆)alkyl, —(C₃-C₆)alkyl,or —(C₃-C₆)cycloalkyl.

In various additional embodiments, R¹ is CH₃, CH₂CH₃, CF₃, CHF₂, CH₂CF₃,CF₂CH₃, or CF₂CF₃. In yet other additional embodiments, G¹ is —OR^(B),and R^(B) is H, CH₃, CH₂CH₃, CF₃, CHF₂, CH₂CF₃, CF₂CH₃, or CF₂CF₃.

In further embodiments, the compound is a compound of Formula II orFormula (III):

In some other embodiments, the compound is a compound of Formula IV:

wherein G¹ is —OR^(B), and R^(B) and R¹ are each independently alkyl orcycloalkyl, wherein the alkyl and cycloalkyl are optionally substitutedwith one or more halo groups.

In various other embodiments, one or more hydrogen atoms is deuterium ortritium, one or more carbon atoms is a carbon isotope, or a combinationthereof.

In yet other embodiments, the compound is any one of compounds (S)- or(R)-2, 4, 6, 8 or 105:

In additional embodiments, the compound is any one of the compoundsshown:

or an enantiomer thereof.

In some additional embodiments, the compound is levorotatory. In otherembodiments, the compound is dextrorotatory. In other embodiments, thecompound (R)-105 or (S)-105. In yet other embodiments, the compound(R)-105.

In other additional embodiments, the compound (an enantiomer orracemate) has a binding affinity for the alpha estrogen receptor (ERα),and the IC₅₀ of the binding affinity is less than about 500 nM. In otherembodiments the IC₅₀ for ERα is about 1 pM to about 1000 nM, about 0.1nM to about 750 nM, about 1 nM to about 250 nM, about 5 nM to about 500nM, or about 10 nM to about 5000 nM. In various other embodiments, thecompound kills or inhibits the growth of cancer cells by hyperactivationof the unfolded protein response (UPR) in the endoplasmic reticulum. Infurther embodiments, the cancer cells are ERα positive cancer cells. Insome other embodiments, the cancer cells are breast cancer cells,ovarian cancer cells, or endometrial cancer cells.

This disclosure also provides a composition comprising the compounddisclosed herein and a second drug. The disclosure further provides apharmaceutical composition comprising the compound in combination with apharmaceutically acceptable diluent, carrier, excipient, or buffer. Insome embodiments of the pharmaceutical composition, the compound is aracemic mixture of (R)-105 and (S)-105. In various embodiments, aracemic mixture of a compound is a mixture of enantiomers wherein themixture of enantiomers has a ratio of about 50:50, about 45:55, about40:60, about 30:70, about 20:80, about 10:90, or about 5:95.

The disclosure additionally provides a method of treating a cancercomprising administering to an ERα positive cancer subject in needthereof a therapeutically effective amount of the compound, therebytreating the cancer in the subject. In further embodiments, the compoundkills or inhibits growth of ERα positive cancer by hyperactivation ofthe unfolded protein response (UPR) in the endoplasmic reticulum. Inother embodiments, the compound is a racemic mixture of (R)-105 and(S)-105. In other embodiments, the ERα positive cancer is a breastcancer, ovarian cancer, uterine cancer, cervical carcinoma, orendometrial cancer.

The disclosure additionally provides use of the compound for thetreatment of an ERα positive disease in a subject in need thereof,wherein a therapeutically effective amount of the compound isadministered to the subject, thereby treating the cancer in the subject.In various embodiments, the ERα positive disease is an ERα positivecancer. In other embodiments, the ERα positive cancer is a breastcancer, ovarian cancer, uterine cancer, cervical carcinoma, orendometrial cancer. In additional embodiments, the compound isadministered orally, by injection, subcutaneously, sublingually,rectally, by infusion, intravenously, by dermal absorption, or through abody cavity or orifice.

This disclosure provides ranges, limits, and deviations to variablessuch as volume, mass, percentages, ratios, etc. It is understood by anordinary person skilled in the art that a range, such as “number1” to“number2”, implies a continuous range of numbers that includes the wholenumbers and fractional numbers. For example, 1 to 10 means 1, 2, 3, 4,5, . . . 9, 10. It also means 1.0, 1.1, 1.2. 1.3, . . . , 9.8, 9.9,10.0, and also means 1.01, 1.02, 1.03, and so on. If the variabledisclosed is a number less than “number10”, it implies a continuousrange that includes whole numbers and fractional numbers less thannumber10, as discussed above. Similarly, if the variable disclosed is anumber greater than “number10”, it implies a continuous range thatincludes whole numbers and fractional numbers greater than number10.These ranges can be modified by the term “about”, whose meaning has beendescribed above.

RESULTS AND DISCUSSION

The CRISPR/Cas9 gene editing system was used to replace wild type ERα inT47D human breast cancer cells with the two most common ERα mutationsseen in metastatic breast cancer, ERαY537S and ERαD538G. The resultingcell lines TYS-4 (also called TYS) and TDG-1 (also called TDG)(T47DERαY537S clone 4 and T47DERαD538G clone 1) exhibit significantresistance to tamoxifen (the active form of tamoxifen isz-4-hydroxytamoxifen; z-OHT) and to fulvestrant/ICI. (Mao et al., 2016).

To allow visualization of tumors harboring these mutations in liveanimal, clonal lines of TYS and TDG cells stably expressing fireflyluciferase were isolated. Orthotopic mouse tumors containing theseTYS-Luc and TYDG-Luc cells are visualized in live animals bybioluminescent imaging (BLI). Because the In Vivo Imaging System (IVIS)has a detection range of more than 10,000-fold and can be used tovisualize progression of both primary tumors and metastatic tumors, BLIusing IVIS is considered the most advanced way to evaluate the efficacyof new anticancer drugs in animal models. No other research team in auniversity, pharmaceutical or biotechnology company has developed celllines combining expression of the breast cancer ERα mutations andluciferase for BLI.

Unbiased high throughput screening was used for small molecules thatblock ERα action to identify novel ERα biomodulators. BHPI was the firstgeneration lead small molecule to emerge from that search. BHPI is apotent first-in-class non-competitive small molecule ERα biomodulatorthat kills therapy-resistant ERα positive breast and endometrial cancercells and blocks growth of ovarian cancer cells. BHPI binds at adifferent site on ERα than tamoxifen and fulvestrant and has a differentmechanism of action. It was demonstrated that BHPI works through ERα toinduce persistent lethal hyperactivation of the anticipatory pathway ofactivation of the unfolded protein response (UPR). In cell culturemodels, BHPI selectively blocks growth, and often killstherapy-resistant, breast, ovarian and endometrial cancer cells.Notably, BHPI blocked proliferation and of the TYS and TDG cellsexpressing ERα mutations identified in metastatic breast cancer.

In a mouse xenograft model of ERα positive breast cancer, at reasonabledoses, BHPI stopped tumor growth and induced rapid and substantial tumorregression.

In xenograft studies using TYS-Luc and TDG-Luc cells after 4 weeks thevehicle control breast tumors had roughly quadrupled in cells. Incontrast, the tumors in mice treated with BHPI exhibited 97%-99.5%regression.

In an orthotopic ovarian cancer xenograft model using OVCAR-3 cells,that are highly resistant to diverse anticancer drugs, the taxanepaclitaxel was ineffective. BHPI alone strongly reduced tumor growth.Notably, tumors were undetectable in mice treated with BHPI pluspaclitaxel and levels of the circulating cancer biomarker CA125progressively declined to undetectable. In both studies, BHPI was welltolerated by the mice.

New Compounds with Superior Ability to Kill Therapy-Resistant BreastCancer Cells

Through synthesis and evaluation, novel compounds that are superior toBHPI in their ability to kill therapy resistant breast cancer cells wereidentified. Compared to BHPI, the lead compound in this group, C-105exhibits superior potency and efficacy.

Assays were developed for compounds with an improved ability to killcancer cells. One of the assays is based on the classical criterion forcell death, loss of membrane integrity as measured by uptake of the dyeTrypan Blue. This is an instrument-based assay that determines thepercentage of cells in a population that have taken up Trypan Blue. Allcells that have taken up Trypan Blue are dead. This novel assay isunique to the disclosed screening workflow. Although Trypan Blue uptakeis universally accepted as a measure of cell death, it has not been usedby others to test potential anticancer drugs. Additional assays used toevaluate cell death include fluorescence activated cell sorting (FACS)and assays based on inhibition of proliferation and determination ofcell number, sometimes in conjunction with raptinal, a compound known toinduce 100% cell death.

Through synthesis, small molecules were obtained that contain a novelstructural feature that imparts surprising results. Compared to BHPI,the lead small molecule, C-105, exhibits far superior potency andefficacy. Notably, unlike BHPI, the active (−) enantiomer of C-105killed 100% of TYS-Luc cells in a long-term cell culture experiment.

Moreover, current endocrine therapy drugs tamoxifen and fulvestrant arecytostatic and showed no ability at all to kill TYS and TDG breastcancer cells. Demonstrating target specificity, even at concentrationsmore than 10 times higher than those that effectively kill ERα positivebreast cancer cells, C-105 had no effect in several ERα negative celllines. Also, the inactive (+) enantiomer was ineffective and not toxicin ERα positive and ERα negative cell lines.

Notably, in a mouse xenograft, after administration of active (−)C-105for just 3 days, the tumors were destroyed. Using BLI to visualize thetumors, 4 of the 5 tumors shrank (regressed) by more than 99.9% and allfive shrank by more than 99%. Using calipers, within seven days, all 5tumors had shrunk to undetectable size. After three weeks, the tumor inone mouse entirely disappeared (compete regression −100%) and the tumorsin 3 of the remaining 4 mice shrank by more than 99.9%. In long durationtest of whether the remaining signals are due to dead or dormant tumorcells, or whether the tumor cells will regrow, after stopping treatmentand waiting 4 weeks, the one tumor that completely disappeared (completeregression −100%) remained at −100% and there was NO “MICRO-TUMOR”REGROWTH in the other mice. This is an unprecedented response.

Mechanism of Action of −105: The actions of −105 include, but are notlimited to, inducing lethal hyperactivation of the endoplasmic reticulumstress sensor, the unfolded protein response (UPR). The endoplasmicreticulum (EnR) stress sensor the unfolded protein response (UPR)balances the synthesis of new proteins with the availability ofchaperones and other proteins that help fold and transport proteinswithin cells. The anticipatory UPR pathway is activated in the absenceof unfolded proteins and anticipates future needs for new proteinfolding capacity (FIG. 32).

To induce lethal hyperactivation of the unfolded protein response −105binds to ERα in cancer cells. This leads to activation of phospholipaseC γ (PLCγ), Activated PLCγ enzymatically produces inositol triphosphate(IP₃). The IP₃ binds to and opens endoplasmic reticulum IP₃ receptorcalcium channels in the EnR. Opening the IP₃R calcium channels resultsin very rapid efflux of calcium stored in the lumen (interior) of theendoplasmic reticulum into the cell body. This hyperactivates the UPR.When activated, one arm of the UPR, the PERK arm, inhibits proteinsynthesis. Activation of another arm of the UPR, IRE1α, inducesformation of the active spliced from of the mRNA encoding thetranscription factor XBP-1 (spXBP-1). To restore calcium homeostasis,powerful SERCA pumps in the membrane of the endoplasmic reticulum carryout ATP dependent pumping of calcium from the cell body into theinterior of the EnR. Because the IP₃R calcium channels remain open, thecalcium pumped into the lumen of the EnR leaks back out. This creates afutile cycle that depletes intracellular ATP.

UPR markers and inhibitors: Formation of spXBP-1 mRNA is used as amarker for UPR activation. The widely used small molecule 2-APB locksthe IP₃Rs closed and prevents the calcium efflux and UPRhyperactivation. The small molecule thapsigargin (THG) potently inhibitsthe SERCA pumps and prevents the cell from using up its ATP stores.

In a treatment and vehicle-controlled mouse tumor study, human breastcancer cells were engineered to contain both the lethal ERαY537Smutation found in metastatic breast cancer, and the gene for fireflyluciferase—when the appropriate chemical is added, the fireflyluciferase breaks down the chemical and light is produced. Using asensitive detector called an imaging system, this allows us to image thetumors inside live mice. This is called bioluminescent imaging, or BLI.Large tumors were allowed to form. Then (−)-105 was injected daily underthe skin for three weeks. Using the imaging system, the effect of(−)-105 on the tumors was monitored. At the same time the effect of thevehicle in which (−)-105 was dissolved on tumors was also monitored.These tumors were treated identically, except that they did not receivethe test drug (−)-105.

The image in FIG. 17 shows one of the vehicle injected control tumors atthe end of the study in a side-by-side comparison with 4 of the 5 micetreated with (−)-105 (the imager can only photograph 5 mice at once).Table 1 shows the light emitted by the tumors. In a mouse xenograft, (−)105 induces rapid and profound regression of large therapy-resistantbreast tumors. Even though tumors in FIG. 17 cannot be seen visually, bygreatly increasing the exposure time and sensitivity the tiny numbers oflight emitting cells in the treated mice can be imaged (FIG. 34).

A study was performed to determine what happens after stopping treatmentwith (−)-105 (Table 2). The study was designed to show whether themicro-tumors would remain dormant, or whether they would regrow. At theend of the three-week treatment, there is residual material that givesoff a tiny amount of light which are termed “micro-tumors”. Based onsensitivity of the imaging system, it is believed these micro-tumorscontain anywhere from 0-˜1,000 cancer cells; the micro-tumors will notbe visible to the naked eye. Seeing them requires the powerful imagingsystem, or a microscope. To test whether these cells are either dead, orno longer dividing and are dormant, the cells two and 4 weeks after theystopped receiving any treatment were imaged. This study tested whethergrowing cancer cells have been eradicated; this study continues.

Bioluminescent imaging (BLI) using luciferase shows near eradication oftherapy-resistant breast tumors in mice in side-by-side imaging of onevehicle injected control mouse and 4 of 5 (−)-105-treated mice (FIG.17). In a 3-week study, large breast tumors were present at start oftreatment and regressed as shown in the image of FIG. 17. The study wasperformed using Orthotopic TYS-Luc (injected cells: T47DERαY537-Luchuman breast cancer cells), e.g., the most lethal ERα mutation in humanin metastatic breast cancer. Tumors were grown in NSG mice. Furtherdetails are provided in the Examples.

TABLE 1 Evaluation of (−)-105 in Orthotopic Mouse Xenograft Tumors UsingBioluminescent Imaging (BLI) of Breast Tumors Expressing the LethalERαY537S Mutation Seen in Metastatic Breast Cancer. Day 0 Day 3 Day 7Day 14 Day 21 Flux Flux Day 3 Flux Day 7 Flux Day 14 Flux Day 21(millions of (millions of % Change in (millions of % Change in (millionsof % Change in (millions of % Change in Group Mouse photons/sec)photons/s) Tumor Flux photons/s) Tumor Flux photons/s) Tumor Fluxphotons/s) Tumor Flux Vehicle 126 182 356 +196 168 −8 270 +148 451 +247Vehicle 146 409 462 +113 338 −17 374 −9 382 −7 Vehicle 148 49.7 159 +318122 +244 116 +232 348 +700 Vehicle 149 201 314 +157 238 +118 296 +147206 +102 Vehicle 150 1,250 2,320 +186 3,040 +243 2,710 +217 3,460 +277Day 0 Day 3 Day 7 Day 14 Day 21 Flux Flux Day 3 Flux Day 7 Flux Day 14Flux Day 21 (millions of (millions of % Change in (millions of % Changein (millions of % Change in (millions of % Change in Group Mousephotons/sec) photons/s) Tumor Flux photons/s) Tumor Flux photons/s)Tumor Flux photons/s) Tumor Flux (−)-105 127 2,130 0.22 −99.99 0.082−99.996 0.246 −99.99 0.051 −99.998 (−)-105 128 277 0.043 −99.98 0.041−99.99 0.039 −99.99 0.16 −99.94 (−)-105 131 72.6 0.40 −99.45 0.17 −99.770.245 −99.66 0.14 −99.81 (−)-105 134 1,880 0.29 −99.98 0.059 −99.996 0−100 0 −100 (−)-105 147 1,650 0.17 −99.99 0.18 −99.99 0.071 −99.9960.046 −99.997The imaging system used for the study is more sensitive and able to pickup tiny numbers of tumor cells than standard measuring systems, such ascalipers that measure tumor size, or weighing the tumors afterdissecting them out of the mice.

TABLE 2 No regrowth of microtumors 4 weeks after (−)-105 treatmentstopped. Week 2 Week 4 Start Recovery Start No No Drug Week 2 No DrugWeek 4 Flux Drug: Tumor Flux No Drug Tumor Flux No Drug (millions of %change in (millions of % Change in (millions of % Change in Group Mousephotons/sec) Tumor Flux photons/sec) Tumor Flux photons/sec) Tumor Flux(−)-105 127 0.051 −.99.997 0.17 −99.991 0.038 −99.998 (−)-105 128 0.16−99.94 0.13 −99.95 0.038 −99.98 (−)-105 131 0.14 −99.81 0.11 −99.840.084 −99.88 (−)-105 134 0 −100 0 −100 0 −100 (−)-105 147 0.046 −99.9970 −100 0.048 −99.997

Key findings in studies showed the following:

(a) (−)-105 rapidly and dramatically destroyed the tumors. After onlythree days, the tumors in all 5 mice shrunk, or regressed, by more than99%. Four of the five tumors shrank by more than 99.9%.(b) At the end of the three-week study, there was no detectable tumor inone mouse (the tumor was eradicated; −100%), the tumors in the otherfour mice were nearly eradicated, with two of them very close to thelimit that the imaging system can detect.(c) Two weeks after treatment was stopped, the microtumors had notstarted growing again. Two of the five mice had no detectable tumors(the tumor was eradicated; −100%), two went down very slightly and onewent up very slightly.(d) Overall, the control vehicle-treated tumors continued to grow. Thedramatic tumor regression was observed is therefore due toadministration of the test drug (−)-105.(e) Using calipers to measure tumor size, the tumors in all 5 mice hadshrunk, (regressed) to undetectable after just 3 days of treatment with(−)-105.(f) Overall, the health of the mice treated with (−)-105 was good. Notoxicity.

General Synthetic Methods

The invention also relates to methods of making the compounds andcompositions of the invention. The compounds and compositions can beprepared by any of the applicable techniques of organic synthesis, forexample, the techniques described herein. Many such techniques are wellknown in the art. However, many of the known techniques are elaboratedin Compendium of Organic Synthetic Methods (John Wiley & Sons, NewYork), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T.Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and LeroyWade, 1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade,Jr., 1984; and Vol. 6, Michael B. Smith; as well as standard organicreference texts such as March's Advanced Organic Chemistry: Reactions,Mechanisms, and Structure, 5^(th) Ed. by M. B. Smith and J. March (JohnWiley & Sons, New York, 2001), Comprehensive Organic Synthesis;Selectivity, Strategy & Efficiency in Modern Organic Chemistry, in 9Volumes, Barry M. Trost, Ed.-in-Chief (Pergamon Press, New York, 1993printing)); Advanced Organic Chemistry, Part B: Reactions and Synthesis,Second Edition, Cary and Sundberg (1983); Protecting Groups in OrganicSynthesis, Second Edition, Greene, T. W., and Wutz, P.G.M., John Wiley &Sons, New York; and Comprehensive Organic Transformations, Larock, R.C., Second Edition, John Wiley & Sons, New York (1999).

A number of exemplary methods for the preparation of the compounds ofthe invention are provided below. These methods are intended toillustrate the nature of such preparations are not intended to limit thescope of applicable methods.

Generally, the reaction conditions such as temperature, reaction time,solvents, work-up procedures, and the like, will be those common in theart for the particular reaction to be performed. The cited referencematerial, together with material cited therein, contains detaileddescriptions of such conditions. Typically, the temperatures will be−100° C. to 200° C., solvents will be aprotic or protic depending on theconditions required, and reaction times will be 1 minute to 10 days.Work-up typically consists of quenching any unreacted reagents followedby partition between a water/organic layer system (extraction) andseparation of the layer containing the product.

Oxidation and reduction reactions are typically carried out attemperatures near room temperature (about 20° C.), although for metalhydride reductions frequently the temperature is reduced to 0° C. to−100° C. Heating can also be used when appropriate. Solvents aretypically aprotic for reductions and may be either protic or aprotic foroxidations. Reaction times are adjusted to achieve desired conversions.

Condensation reactions are typically carried out at temperatures nearroom temperature, although for non-equilibrating, kinetically controlledcondensations reduced temperatures (0° C. to −100° C.) are also common.Solvents can be either protic (common in equilibrating reactions) oraprotic (common in kinetically controlled reactions). Standard synthetictechniques such as azeotropic removal of reaction by-products and use ofanhydrous reaction conditions (e.g. inert gas environments) are commonin the art and will be applied when applicable.

Protecting Groups. The term “protecting group” refers to any groupwhich, when bound to a hydroxy or other heteroatom prevents undesiredreactions from occurring at this group and which can be removed byconventional chemical or enzymatic steps to reestablish the hydroxylgroup. The particular removable protecting group employed is not alwayscritical and preferred removable hydroxyl blocking groups includeconventional substituents such as, for example, allyl, benzyl, acetyl,chloroacetyl, thiobenzyl, benzylidene, phenacyl, methyl methoxy, silylethers (e.g., trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), ort-butyldimethylsilyl (TBS)) and any other group that can be introducedchemically onto a hydroxyl functionality and later selectively removedeither by chemical or enzymatic methods in mild conditions compatiblewith the nature of the product.

Suitable hydroxyl protecting groups are known to those skilled in theart and disclosed in more detail in T. W. Greene, Protecting Groups InOrganic Synthesis; Wiley: New York, 1981 (“Greene”) and the referencescited therein, and Kocienski, Philip J.; Protecting Groups (Georg ThiemeVerlag Stuttgart, New York, 1994), both of which are incorporated hereinby reference.

Protecting groups are available, commonly known and used, and areoptionally used to prevent side reactions with the protected groupduring synthetic procedures, i.e. routes or methods to prepare thecompounds by the methods of the invention. For the most part thedecision as to which groups to protect, when to do so, and the nature ofthe chemical protecting group “PG” will be dependent upon the chemistryof the reaction to be protected against (e.g., acidic, basic, oxidative,reductive or other conditions) and the intended direction of thesynthesis.

Schemes 1-3 show the general synthesis of the compounds disclosedherein, such as the compounds in Scheme 4. Detailed experimentalconditions are provided in the Examples.

Basis of Assays for Killing of Cancer Cells.

TRYPAN BLUE: For decades, viable cells have been measured by theirability to exclude the dye Trypan Blue. Cells with intact membranes donot take up Trypan Blue. Dead cells, which have lost membrane integrity,take up the dye and are blue. Thus, the percentage of dead cells is thepercentage of cells that are Trypan Blue positive out of the total cellpopulation. Quantitative assessment of Trypan Blue uptake uses a cellcounter from Fisher. Since the assay is instrument based, there is noobserver bias in determining the percentage of Trypan Blue positivecells.

ALAMAR BLUE/RAPTINAL ASSAY: Live cells maintain a reducing environment.The non-fluorescent cell permeable ingredient of Alamar Blue®(resazurin) is taken up by cells. In living cells, it is reduced to thefluorescent compound resorufin. Using a standard curve of cell numberversus fluorescence the number of live cells in a test sample can bedetermined. At high concentrations raptinal kills 100% of cells. Thevehicle in which the test compound is dissolved is not toxic. Thus,after subtracting the blank for medium alone, the fluorescence readingfor vehicle corresponds to 100% viable cells and the signal for raptinalcorresponds to 0% viable cells. While this assay provides a less directmeasurement of cell death than the trypan blue assay, it is more easilyscaled up to large numbers of samples.

FLOW CYTOMETRY (FITC): Dying cells exhibit specific changes that can bemonitored using a fluorescence activated cell sorter (FACS). One suchchange is loss of plasma membrane polarity. This is monitored usingAnnexin V. Dying cells also take up the dye propidium iodide. Propidiumiodide (PI) fluoresces when it is intercalated into DNA. These assaysmonitor cells at specific stages of cell death. For example, cells thatare dead and whose DNA has been destroyed and is in such small piecesthat it no longer takes up PI are dead but are no longer seen as PIpositive.

Assay Execution and Conditions

TRYPAN BLUE ASSAYS: Assays are performed on cells in 6 well plates:300,000 cells are plated in each well of a 6 well plate in 3 ml ofmedium. After 24 hours, the DMSO vehicle or the indicated compound isadded (in 1000^(th) of the vol.) The cells incubated for an additional24 hours, and then harvested in 0.25 ml of trypsin, followed afterharvest by 0.75 ml of medium. The cells are spun down and resuspended in100-200 μl of medium left that is over the cell pellet (resuspend cellsby pipetting up and down a few times). Add 10 μl to 10 μl of trypanblue. Then 10 μl is inserted into the counting slide. After insertingthe slide into the Fisher TC20 counting is automatic (these many cellsare needed because an accurate ratio of Trypan Blue positive to TrypanBlue negative cells requires about 200 cells in the field the instrumentcounts).

ALAMAR BLUE RAPTINAL ASSAYS: Cells were plated at 4,000-10,000cells/well (96 well plate), usually in 50 μl of medium. After about 18hours, an additional 50 μl of medium containing 2× the desired finalconcentration of the test compound (C-105) was added to each well. After24 hours Alamar Blue reagent was added and fluorescence in the wells wasread (BMG PheraStar or Molecular Devices Spectra Max M3 MicroplateReader). % relative death was determined by setting the reading forraptinal-treated cells to 100% dead cells and the reading forvehicle-treated cells to 0% dead cells.

FLOW CYTOMETRY (FITC) ASSAYS: Cells were seeded in 12-well plates,75,000 cells/well, and allowed to adhere overnight. The next day, theindicated concentrations of raptinal (positive control), (−)-105,(±)-105, and (+)-105 were added and allowed to incubate at 37° C. for 24hours (Final Volume: 1 ml, 0.1% DMSO). After incubation, cells wereharvested and resuspended in 350 μl of cold Annexin binding buffer (10mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂), pH 7.4) premixed with AnnexinV-FITC and PI dyes. Samples were analyzed on a BD Biosciences LSRII flowcytometer, and data analysis was performed using FSC Express Version5-6. Cell death was assessed relative to a DMSO treated control.

Cell Lines Used for the Therapeutic Target: ERα Positive Breast CancerCells

TDG and TDG-LUC (T47DERαD538G clone 1): Both copies of the wild type ERαgene replaced with the ERαD538G mutation seen in metastatic breastcancer. These cells grow without estrogen, with a further modestincrease in growth rate on addition of estrogen. Substantial resistanceto z-OHT (the active form of tamoxifen) and fulvestrant//Faslodex/ICI182,780. Slightly less resistant to z-OHT and fulvestrant/ICI than theERαY537S cells. ERαD538G is the single most common mutation inmetastatic breast cancer. Therefore, these cells are critical fortesting. (TDG-LUC) TDG cells stably transfected to express fireflyluciferase. Transcription of the luciferase gene is driven by aconstitutively active CMV promoter.

TYS and TYS-LUC (T47DERαY537S CLONE 4): Both copies of wild type ERαgene replaced with the ERαY537S mutation seen in metastatic breastcancer. Fast growth without estrogen. No additional growth stimulationby estrogen. Substantial resistance to z-OHT and fulvestrant/ICI.Slightly more resistant to current drugs than the ERαD538G cells. Thisis the most lethal mutation in metastatic breast cancer. (TYS-Luc) TYScells stably transfected to express firefly luciferase. Transcription ofthe luciferase gene is driven by a constitutively active CMV promoter.

T47D: ERα positive, require estrogen to grow, sensitive to z-OHT andfulvestrant/ICI; parental cell line, widely used, but less common thanMCF-7 cells. The lower levels of ERα in T47D cells, compared to MCF-7cells, are more in-line with ERα levels in actual breast cancers. T47Dcells were tested in part because the T47D cells require higherconcentrations of BHPI to inhibit proliferation and kill than the twocell lines expressing mutant ERα.

MCF-7 (Michigan Cancer Foundation-7): ERα positive, the most widely usedERα positive breast cancer cell line. Estrogen greatly stimulates theirgrowth; sensitive to z-OHT and fulvestrant/ICI.

ECC-1: ERα positive uterine cancer cells.

Caov3: Drug resistant ERα positive ovarian cancer cells. BHPI blocksgrowth of Caov-3 cells but does not kill them. These cells arecompletely resistant to tamoxifen and fulvestrant and partiallyresistant to cisplatin and paclitaxel.

ERα Negative Cells Used to Test for Toxicity and Off-Target Effects

HeLa: These are ERα negative cells of cervical origin. The most widelyused human cell line.

MDA-MB-231 cells: These are ERα negative cells and are the most commonmodel for triple negative breast cancer. Highly metastatic. These cellsare used because in this work they are more sensitive to non-specificgrowth inhibition than most other cell lines. They are therefore astringent test for non-specific toxicity.

MCF-10A (Michigan Cancer Foundation-JOA): Immortal, but not tumorigenic,ERα negative breast cell line.

Statistics

Unless otherwise stated there are at least 3 biological replicates ofeach sample (n=3). Data is presented as the average of thereplicates±S.E.M. For statistical significance comparisons are for thecells treated with the vehicle compared to the cells treated with thesame concentration of 105, BHPI, or other compounds, wherein P<0.05; **P<0.01; *** P<0.001 all using Students T-test.

Pharmaceutical Formulations

The compounds described herein can be used to prepare therapeuticpharmaceutical compositions, for example, by combining the compoundswith a pharmaceutically acceptable diluent, excipient, or carrier. Thecompounds may be added to a carrier in the form of a salt or solvate.For example, in cases where compounds are sufficiently basic or acidicto form stable nontoxic acid or base salts, administration of thecompounds as salts may be appropriate. Examples of pharmaceuticallyacceptable salts are organic acid addition salts formed with acids thatform a physiologically acceptable anion, for example, tosylate,methanesulfonate, acetate, citrate, malonate, tartrate, succinate,benzoate, ascorbate, α-ketoglutarate, and β-glycerophosphate. Suitableinorganic salts may also be formed, including hydrochloride, halide,sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid to provide aphysiologically acceptable ionic compound. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for example,calcium) salts of carboxylic acids can also be prepared by analogousmethods.

The compounds of the formulas described herein can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient, in a variety of forms. The forms can be specificallyadapted to a chosen route of administration, e.g., oral or parenteraladministration, by intravenous, intramuscular, topical or subcutaneousroutes.

The compounds described herein may be systemically administered incombination with a pharmaceutically acceptable vehicle, such as an inertdiluent or an assimilable edible carrier. For oral administration,compounds can be enclosed in hard or soft-shell gelatin capsules,compressed into tablets, or incorporated directly into the food of apatient's diet. Compounds may also be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations typically contain at least 0.1% ofactive compound. The percentage of the compositions and preparations canvary and may conveniently be from about 0.5% to about 60%, about 1% toabout 25%, or about 2% to about 10%, of the weight of a given unitdosage form. The amount of active compound in such therapeuticallyuseful compositions can be such that an effective dosage level can beobtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye andflavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

The active compound may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can be prepared in glycerol, liquidpolyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and/or antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, optionally followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thesolution.

For topical administration, compounds may be applied in pure form, e.g.,when they are liquids. However, it will generally be desirable toadminister the active agent to the skin as a composition or formulation,for example, in combination with a dermatologically acceptable carrier,which may be a solid, a liquid, a gel, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina, and the like. Useful liquidcarriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, orwater-alcohol/glycol blends, in which a compound can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using a pump-type or aerosol sprayer.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses, or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of dermatological compositions for delivering active agents tothe skin are known to the art; for example, see U.S. Pat. No. 4,992,478(Geria), U.S. Pat. No. 4,820,508 (Wortzman), U.S. Pat. No. 4,608,392(Jacquet et al.), and U.S. Pat. No. 4,559,157 (Smith et al.). Suchdermatological compositions can be used in combinations with thecompounds described herein where an ingredient of such compositions canoptionally be replaced by a compound described herein, or a compounddescribed herein can be added to the composition.

Useful dosages of the compounds described herein can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of a compound, or anactive salt or derivative thereof, required for use in treatment willvary not only with the particular compound or salt selected but alsowith the route of administration, the nature of the condition beingtreated, and the age and condition of the patient, and will beultimately at the discretion of an attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently formulated in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form. Inone embodiment, the invention provides a composition comprising acompound of the invention formulated in such a unit dosage form.

The compound can be conveniently administered in a unit dosage form, forexample, containing 5 to 1000 mg/m², conveniently 10 to 750 mg/m², mostconveniently, 50 to 500 mg/m² of active ingredient per unit dosage form.The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The compounds described herein can be effective anti-tumor agents andhave higher potency and/or reduced toxicity as compared to BHPI.Preferably, compounds of the invention are more potent and less toxicthan BHPI, and/or avoid a potential site of catabolic metabolismencountered with BHPI, i.e., have a different metabolic profile thanBHPI.

The invention provides therapeutic methods of treating cancer in amammal, which involve administering to a mammal having cancer aneffective amount of a compound or composition described herein. A mammalincludes a primate, human, rodent, canine, feline, bovine, ovine,equine, swine, caprine, bovine and the like. Cancer refers to anyvarious type of malignant neoplasm, for example, colon cancer, breastcancer, melanoma and leukemia, and in general is characterized by anundesirable cellular proliferation, e.g., unregulated growth, lack ofdifferentiation, local tissue invasion, and metastasis.

The ability of a compound of the invention to treat cancer may bedetermined by using assays well known to the art. For example, thedesign of treatment protocols, toxicity evaluation, data analysis,quantification of tumor cell kills, and the biological significance ofthe use of transplantable tumor screens are known. In addition, abilityof a compound to treat cancer may be determined using the Tests asdescribed below.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES Example 1. Synthetic Procedures

General Information: Unless otherwise stated, all reagents werepurchased from commercial sources and used without further drying orpurification. Solvents used herein were dried after being passed throughactivated alumina columns. All reactions were run in flame-driedglassware under a positive pressure of nitrogen gas. ¹H NMR and ¹³C NMRexperiments were conducted on a Bruker cryoprobe at 500 MHz and 188 MHzrespectively. Spectra obtained in CD₃OD were referenced for 3.31 ppm and49.00 ppm for ¹H and ¹³C NMR spectra respectively. NMR multiplicitiesare reported as: s=singlet, d=doublet, t=triplet, q=quartet,m=multiplet. ¹³C multiplicities are all singlets unless otherwise noted.

X   X Y 7-methylisatin CH₃   1 CH₃ H 7-(trifluoromethyl)isatin CF₃   3CF₃ H

Procedure A: A round bottom flask was charged with desired phenylbromide (3.08 mmol) and dissolved in THF (3.0 mL). The reaction mixturewas cooled to −78° C. and a solution of n-BuLi (2.77 mmol, 1.7 mL) addeddropwise over 10 minutes. The reaction was stirred for 1 hour. Inanother flask, the desired isatin (1.54 mmol) was added and dissolved inTHF (9.4 mL). This solution of isatin was added to the reaction vesseldropwise over 10 minutes. The resultant mixture was stirred at −78° C.for 1 hour, warmed to r.t., and then stirred for 1 hour. The reactionwas quenched with water (10 mL). The solution was extracted with ethylacetate (3×) and the combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The resulting oil was runthrough a silica plug (elution solvent gradient: 10% EtOAc/Hexanesramped to 100% EtOAc). The eluted species was then charged to a roundbottom flask, purged of air, placed under nitrogen atmosphere, anddissolved in THF (15.4 mL). A solution of tetra-n-butylammonium fluoride(5.4 mL) was added and the reaction vessel. After 16 hours of stirring,the reaction was quenched with a 1:1 solution of saturated ammoniumchloride_(aq):water (30 mL). The solution was extracted with ethylacetate (3×) and the combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The resultant oil was thenpurified via column chromatography.

X   X 7-methylisatin CH₃   5 CH₃

Procedure B: A round bottom flask was charged with desired phenylbromide (3.35 mmol) and dissolved in THF (2.8 mL). The reaction mixturewas cooled to −78° C. and a solution of n-BuLi (2.79 mmol, 1.8 mL) addeddropwise over 10 minutes. The reaction was stirred for 1 hour. Inanother flask, the desired isatin (1.86 mmol) was added and dissolved inTHF (5.6 mL). This solution of isatin was added to the reaction vesseldropwise over 10 minutes. The resultant mixture was stirred at −78° C.for 1 hour, warmed to r.t., and then stirred for 1 hour. The reactionwas quenched with water (10 mL). The solution was extracted with ethylacetate (3×) and the combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The resulting oil was runthrough a silica plug (elution solvent gradient: 10% EtOAc/Hexanesramped to 100% EtOAc).

X Y   X Y R 1 CH₃ H   2 CH₃ H CH₃ 3 CF₃ H   4 CF₃ H CH₃ 5 CH₃ CF₃   6CH₃ CF₃ H

Procedure C: A round bottom flask was charged with desired tertiaryalcohol (0.93 mmol) and desired phenol (4.2 mmol) and dissolved indichloromethane (4.7 mL). The reaction mixture was then placed in an icebath and triflic acid (TfOH, 0.42 mL) was then added dropwise. Thereaction vessel was removed from the ice bath and stirred at roomtemperature for 1 hour. The reaction mixture was then poured into anice-filled sodium bicarbonate and the aqueous solution was extractedwith ethyl acetate (3×). The combined organic layers were dried oversodium sulfate, filtered, and concentrated in vacuo. The resultant oilwas then purified via column chromatography.

3-hydroxy-3-(4-hydroxyphenyl)-7-methylindolin-2-one (1): UtilizingGeneral Procedure A, 1 was isolated in 45% yield over two steps. ¹H NMR(CD₃OD, 500 MHz): δ 7.19 (d, J=8.86 Hz 2H), 7.10 (d, J=7.59 Hz 1H), 7.02(d, J=7.46 Hz 1H), 6.96 (dd, J=7.54 Hz, 7.49 Hz 1H), 6.71 (d, J=8.72 Hz2H), 2.29 (s, 3H).

¹³C NMR (CD₃OD, 188 MHz): δ 182.20, 158.40, 141.39, 134.47, 132.71,131.78, 128.23, 123.86, 123.48, 121.00, 115.93, 79.17, 16.58.

3-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-7-methylindolin-2-one (2):Utilizing General Procedure C, 2 was isolated in 83% yield. ¹H NMR(CD₃OD, 500 MHz) δ: 7.12 (d, J=8.93 Hz, 2H), 7.02 (m, 3H), 6.94 (m, 2H),6.82 (d, J=6.82 Hz, 2H), 6.69 (d, J=8.74 Hz), 3.74 (s, 3H), 2.30 (s,3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 182.87, 160.24, 157.71, 140.70, 135.73,135.65, 134.26, 130.58, 130.54, 130.38, 124.48, 123.51, 121.02, 116.02,114.64, 63.36, 55.67, 16.81.

HRMS (ESI): m/z calc. for C₂₂H₂₀NO₃ [M+H]⁺ 346.1443, found: 346.1442.

3-hydroxy-3-(4-hydroxyphenyl)-7-(trifluoromethyl)indolin-2-one (3):Utilizing General Procedure A, 3 was isolated in 35% yield over twosteps. ¹H NMR (CD₃OD, 500 MHz) δ: 7.54 (m, 1H), 7.43 (d, J=7.49 Hz 1H),7.19 (m, 3H), 6.74 (d, J=8.74 Hz, 2H). ¹³C NMR (CD₃OD, 188 MHz) δ:181.60, 158.73, 140.54 (q, J=2.25 Hz), 136.85, 131.80, 129.89, 128.89,126.90 (q, J=4.6 Hz), 125.16 (q, J=270.76 Hz), 123.74, 116.16, 113.56(q, J=33.17 Hz), 77.66.

3-(4-hydroxyphenyl)-3-(4-methoxyphenyl)-7-(trifluoromethyl)indolin-2-one(4): Utilizing General Procedure C, 4 was isolated in 82% yield. ¹H NMR(CD₃OD, 500 MHz) δ: 7.50 (dd, J=8.09 Hz, 0.62 Hz, 1H), 7.40 (dd, J=7.76Hz, 0.64 Hz, 1H), 7.18 (ddd, J=8.08 Hz, 7.46 Hz, 0.87 Hz, 1H), 7.12 (d,J=8.87 Hz, 2H), 7.01 (d, J=8.76 Hz, 2H), 6.86 (d, J=8.87 Hz, 2H), 6.72(d, J=8.75 Hz 2H), 3.77 (s, 3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 182.24, 160.55, 158.10, 139.74 (m), 137.85,134.73, 133.30, 130.98, 130.50, 130.49, 125.72 (q, J=4.6 Hz, 125.23 (q,J=270.71 Hz), 123.32, 116.27, 114.89, 113.55 (q, J=33.50 Hz), 62.12,55.72.

HRMS (ESI): m/z calc. for C₂₂H₁₇NO₃F₃ [M+H]⁺ 400.1161, found: 400.1154.

3-hydroxy-7-methyl-3-(4-(trifluoromethoxy)phenyl)indolin-2-one (5):Utilizing General Procedure B, 5 was isolated in 68% yield over twosteps. ¹H NMR (CD₃OD, 500 MHz) δ: 7.46 (d, J=8.83 Hz 2H), 7.22 (dd,J=1.02 Hz, 8.98 Hz, 2H), 7.13 (m, 1H), 6.98 (m, 2H), 2.31 (s, 3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 181.41, 150.1 (q, J=1.6 Hz), 141.55, 141.46,134.03, 132.19, 128.67, 124.13, 123.4, 121.90 (q, J=255.61 Hz), 121.32,79.04, 16.61.

3-(4-hydroxyphenyl)-7-methyl-3-(4-(trifluoromethoxy)phenyl)indolin-2-one(6): Utilizing General Procedure C, 6 was isolated in 59% yield. ¹H NMR(CD₃OD, 500 MHz) δ: 7.30 (d, J=8.86 Hz 2H), 7.20 (m, 2H), 7.08 (m, 1H),7.03 (d, J=8.74 Hz, 2H), 6.99 (m, 2H), 6.72 (d, J=8.73 Hz, 2H), 2.31 (s,3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 182.00, 158.02, 149.56 (q, J=1.47 Hz),143.12, 140.82, 134.85, 133.55, 131.22, 130.77, 130.56, 124.51, 123.73,121.89 (q, J=255.61 Hz), 121.78, 121.32, 116.23, 63.49, 16.82.

HRMS (ESI): m/z calc. for C₂₂H₁₇NO₃F₃ [M+H]⁺ 400.1161, found: 400.1163.

Synthesis of3-(4-hydroxyphenyl)-3-(4-(trifluoromethoxy)phenyl)-7-(trifluoromethyl)indolin-2-one((±)-105): A round bottom flask was charged with1-bromo-4-(trifluoromethoxy)benzene (3.08 mmol) and dissolved in THF(3.0 mL). The reaction mixture was cooled to −78° C. and a solution ofn-BuLi (2.77 mmol, 1.7 mL) added dropwise over 10 minutes. The reactionwas stirred for 1 hour. In another flask, the desired isatin (1.54 mmol)was added and dissolved in THF (9.4 mL). This solution of isatin wasadded to the reaction vessel dropwise over 10 minutes. The resultantmixture was stirred at −78° C. for 1 hour, warmed to r.t., and thenstirred for 1 hour. The reaction was quenched with water (10 mL). Thesolution was extracted with ethyl acetate (3×) and the combined organiclayers were dried over sodium sulfate, filtered, and concentrated invacuo. A new round bottom flask was charged with crude tertiary alcoholand phenol (6.95 mmol) and dissolved in dichloromethane (7.7 mL). Thereaction mixture was then placed in an ice bath and triflic acid (TfOH,0.7 mL) was then added dropwise. The reaction vessel was removed fromthe ice bath and stirred at room temperature for 1 hour. The reactionmixture was then poured into an ice-filled sodium bicarbonate and theaqueous solution was extracted with ethyl acetate (3×). The combinedorganic layers were dried over sodium sulfate, filtered, andconcentrated in vacuo. The resultant oil was then purified via columnchromatography. (±)-105 was isolated in 50% yield over two syntheticsteps.

¹H NMR (CD₃OD, 500 MHz) δ: 7.54 (d, J=7.95 Hz, 1H), 7.45 (d, J=7.48 Hz,1H), 7.31 (d, J=8.91 Hz 2H), 7.22 (m, 3H), 7.03 (d, J=8.85 Hz, 2H), 6.75(d, J=8.80 Hz, 2H).

¹³C NMR (CD₃OD, 188 MHz) δ: 181.35, 158.37, 149.81 (q, J=1.78 Hz),142.17, 139.84 (q, J=2.29 Hz), 136.92, 132.65, 131.22, 131.06, 130.48,126.12 (q, J=4.58 Hz), 125.15 (q, J=270.89 Hz), 123.55, 121.88 (q,J=255.84 Hz), 122.01, 116.48, 113.77 (q, J=33.32 Hz), 62.22.

HRMS (ESI): m/z calc. for C₂₂H₁₄NO₃F₆ [M+H]⁺ 454.0878, found: 454.0878.

Chiral Separation of (±)-105: (±)-105 was separated into its respectiveenantiomers using preparative chiral HPLC separation (Lux® 5 μMCellulose-1, LC Column, 250×21.2 mm, AXIA™ Packed, isocratic: 7%i-PrOH/hexanes). Collected fractions yielded (−)-105 (peak A) and(+)-105 (peak B): FIG. 5. This method of chiral separation yields(−)-105 as a white solid (enantiomeric purity=>99%,⁵⁸⁹[α]_(CHCl3)=−14.6° (±)_(0.1)°) and (+)-105 as a white solid(enantiomeric purity=˜98-99%, ⁵⁸⁹[α]_(CHCl3)=−13.8° (±))_(0.1)° (seeExample 4). Enantiopurity determination was conducted by analyticalchiral HPLC column: CHIRALPAK® IB-3 (particle size 3 μM, dimensions: 4.6mm×100 mm, separation method, isochratic 7.5% i-PrOH/hexanes) (FIG.6-8).

6-chloro-3-hydroxy-7-methyl-3-(4-(trifluoromethoxy)phenyl)indolin-2-one(7): A reaction vessel was charged with substituted aniline (2.82 mmol),1M HCl (2.82 mL), water (18.8 mL), anhydrous sodium sulfate (16.92mmol), and hydroxylamine hydrochloride (9.17 mmol). The mixture washeated to boiling and then chloral hydrate was added as one portion. Thereaction was kept at reflux for 40 minutes, then cooled to reflux andthe aqueous solution was extracted with ethyl acetate (3×). The combinedorganic layers were dried over sodium sulfate, filtered, andconcentrated in vacuo. Concentrated sulfuric acid (3 mL) was then addedto the resultant residue. This solution was heated to 80° C. for 20minutes, then poured onto ice. The resulting aqueous mixture wasextracted with ethyl acetate (3×), and the combined organic layers driedover sodium sulfate, filtered, and concentrated in vacuo. The red-brownsolid obtained after concentration proved to be poorly soluble in mostorganic solvents.

To a new reaction vessel, the desired phenyl bromide (3.08 mmol) anddissolved in THF (3.0 mL). The reaction mixture was cooled to −78° C.and a solution of n-BuLi (2.77 mmol, 1.73 mL) added dropwise over 10minutes. The reaction was stirred for 1 hour. In another flask,6-chloro-7methylisatin (1.54 mmol) was added and dissolved in THF (9.4mL). This solution of isatin was added to the reaction vessel dropwiseover 10 minutes. The resultant mixture was stirred at −78° C. for 1hour, warmed to r.t., and then stirred for 1 hour. The reaction wasquenched with water (10 mL). The solution was extracted with ethylacetate (3×) and the combined organic layers were dried over sodiumsulfate, filtered, and concentrated in vacuo. The resulting oil was runthrough a silica plug (elution solvent gradient: 10% EtOAc/Hexanesramped to 100% EtOAc).

7 was isolated in 24% yield over three synthetic steps. ¹H NMR (CD₃OD,500 MHz) δ: 7.46 (d, J=8.85 Hz 2H), 7.23 (d, J=7.98 Hz, 2H), 7.10 (d,J=7.97 Hz 1H), 6.98 (d, J=8.02 Hz, 1H), 2.35 (s, 3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 181.25, 150.17 (q, J=1.83 Hz), 143.14,140.98, 136.68, 132.74, 128.63, 124.53, 124.40, 121.88 (q, J=255.75 Hz),121.86, 119.76, 78.91, 14.11.

HRMS (ESI): m/z calc. for C₁₆H₁₁NO₃F₃ClNa [M+Na]⁺380.0277, found:380.0284.

6-chloro-3-(4-hydroxyphenyl)-7-methyl-3-(4-(trifluoromethoxy)phenyl)indolin-2-one(8): Utilizing General Procedure C, 8 was isolated in 85% yield. ¹H NMR(CD₃OD, 500 MHz) δ: 7.30 (d, J=8.91 Hz 2H), 7.21 (m, 2H), 7.10 (d,J=8.10 Hz, 1H), 7.02 (m, 4H), 6.73 (d, J=8.80 Hz, 2H), 2.35 (s, 3H).

¹³C NMR (CD₃OD, 188 MHz) δ: 181.81, 158.16, 149.67 (q, J=2.02 Hz),142.6, 142.38, 135.34, 133.50, 133.10, 131.18, 130.47, 125.48, 124.12,121.89 (q, J=255.49 Hz), 121.8, 119.66, 116.35, 63.45, 14.30.

HRMS (ESI): m/z calc. for C₂₂H₁₆NO₃F₃Cl [M+H]⁺ 434.0771, found:434.0764.

Separation of enantiomers of 105: Racemic mixtures of 105 were separatedinto respective enantiomers (FIG. 5) using preparative chiral HPLCseparation (Lux® 5 Cellulose-1, LC Column, 250×21.2 mm, AXIA™ Packed,isocratic: 7% i-PrOH/hexanes). Collected fractions yielded (−)-105 (peakA) and (+)-105 (peak B) This method of chiral separation yields (−)-105as a white solid (enantiomeric purity=>99%, [α]=−134) and (+)-105 as awhite solid (enantiomeric purity=98->99%, [α]=not determined).Enantiopurity determination was conducted by analytical chiral HPLCcolumn: CHIRALPAK® IB-3 (particle size 3 dimensions: 4.6 mm×100 mm,separation method, isochratic 7.5% i-PrOH/hexanes). The compound (−)-105bends light in the negative direction ([α] value). FIG. 6 is aquantitative chiral HPLC trace showing the separated peaks of (±)-105that are present in about equal concentrations. FIG. 7 shows the active(−)-105 peak is highly pure after separation. FIG. 8 shows the inactive(+)-105 peak is also pure after separation. Because there is a trace ofthe active (−)-105 in this preparation, and because of the outstandingpotency of (−)-105, at extremely high concentrations (+)-105 may exhibitsome activity.

Example 2. Cancer Cell Death Studies of Compound 105

Experimental Details of the Xenografts: TYS-Luc cells (5,000,000 cellsin Matrigel) were injected into the upper mammary fat pad ofovariectomized female NSG (SCID) mice (Jackson labs). After 10 weeks, onday 0, large tumors (size range: 100-600 mm³) were present. Mice weretreated with the vehicle used to dissolve (−)-105, or (−)-105 at 40mg/kd daily by subcutaneous injection. For bioluminescent imaging (BLI)of the tumors, the mice were anesthetized with isoflurane and Lucerfin,the luciferase substrate, was injected into the mice. Flux, the totalmember of light photons hitting the detector in one second was measuredusing an IVIS (In Vitro Imaging System). Shown is the flux (in millionsof units per second) for the entire area containing each tumor, or anequivalent area when the tumor is tiny or undetectable and the percentchange in tumor size at each time a measurement was taken (day 0, day 3,week 1, week 2, week 3). Then treatment was stopped, and anothermeasurement was taken two weeks letter. Note that tumors were alsomeasured with calipers. Since some tumors do not protrude much, this isfar less accurate than BLI. After three days all the (−)-105 tumors hasdeclined to undetectable using calipers.

FIG. 1 shows the dose response effect of (±)-105 on ERα positive and ERαnegative human cancer cells, demonstrating anticancer activity. ERαpositive MCF-7 breast cancer, Caov-3 ovarian cancer and ECC-1 uterinecancer cells and ERα negative MDA-MB-231 human breast cancer cells andHeLa, human cervical carcinoma cells and MCF-10A human mammaryepithelial cells were plated at 10,000 cells/well (96 well plate) in 50μl of medium. After about 18 hours, an additional 50 μl of mediumcontaining 2× the desired final concentration of the test compound(C-105) was added to each well (final vol. 100 μl/well). After 24 hoursAlamar Blue reagent was added and fluorescence in the wells was readafter 1 hour (BMG PheraStar Microplate Reader). % relative death wasdetermined by setting the reading for 10,000 nM raptinal-treated cellsto 100% dead cells and the reading for vehicle-treated cells to 0% deadcells. (Data is the average±SEM of 3 independent experiments with 5biological replicates for each sample within each independentexperiment.)

FIG. 2 shows the dose response comparing the ability of BHPI and (±)-105to kill ERα positive T47D cells. T47D, human breast cancer cells wereplated at 75,000 cells/well. The next day, DMSO vehicle, 10 μm raptinal(control for 100% cell death), or 0, 50, 100 500 and 1,000 nM of BHPI or(±)-105, or 1,000 nM ICI/fulvestrant or 1,000 nM OHT was added. After 24hours viable cells were determined by FACS using V-FITC and propidiumiodide dyes. (n=3 independent experiments±SEM). Results from this flowcytometry experiment (that detects dead cells) show that (±)-105 iscytotoxic and not cytostatic. Whereas compounds OHT, ICI and BHPI areall cytostatic based on low observed activity. The observed activity of(±)-105 in this assay is similar to the positive control, raptinal, acompound that also quantitatively kills cancer cells (see also FIG.36A).

FIG. 3 compares the ability of (±)-105 to kill ERα positive and ERαnegative cells. The dose response tests the ability of (±)-105 torapidly kill ERα positive and ERα negative cells. Cell lines: ERαpositive breast cancer: T47D, MCF-1 TYS (Y537S); Endometrial: ECC-1; ERαnegative cell lines: Breast MDA-MB-231; Melanoma: OMM1, A375; Lung:H3122; Colon: HT-29; fibroblast: HFF-1. 4,000-8,000 cells/well wereplated. The next day (±)-105 at concentrations from 0-10,000 nM wasadded. After 24 hours Alamar Blue reagent was added and fluorescence inthe wells was read. % relative death was determined by setting thereading for 1,000 nM raptinal-treated cells to 100% dead cells and thereading for vehicle-treated cells to 0% dead cells. (n=3±SEM). Theseresults show that racemic compound 105 is selective for ER□ positivecancer cell lines; also demonstrated with enantiomerically pure (−)-105in FIG. 36F.

FIG. 4A illustrates how IC₅₀'s for cell death were determined. 4B teststhe ability BHPI, (−)-105, and 01-15 to kill. 4C shows (−)-105 iseffective when administered orally. Large orthotopic breast tumors ofTYD-Luc cells (T47DERαD538G-luciferase human breast cancer cells) wereallowed to form for 4 months in immune suppressed NSG mice. Mice weretreated for 3 days with either vehicle only (Vehicle), oral gavage of 40mg/kg (−)-105 (oral), or daily injection of 40 mg/kg (−)-105 (injected).Tumors were visualized by bioluminescent imaging using luciferase. Thedashed line represents −100%; tumor regression to undetectable. Thus,the result show (−)-105 is superior to BHPI and 01-15.

FIG. 9 shows that the (−) enantiomer of 105 is active and the (+)enantiomer is inactive. 75,000 ERα positive T47D, human breast cancercells, were plated/well. The next day, DMSO vehicle, 10 μm raptinal(control for 100% cell death), or 100 500 and 1,000 nM of (+)-105, or(−)-105. After 24 hours viable cells were determined by FACS usingV-FITC and propidium iodide dyes. (n=1).

FIG. 10 shows that (−)-105 induces dose-dependent death of MCF-7 cells;(+)-105 is inactive. 75,000 ERα positive T47D, human breast cancercells, were plated/well. The next day, DMSO vehicle, 10 μm raptinal(control for 100% cell death), or 1-500 nM of (−)-105 or 1,000 nM(+)-105 or 1,000 nM (±)-105 were added. After 24 hours viable cells weredetermined by FACS using V-FITC and propidium iodide dyes. (n=3independent experiments±SEM).

FIG. 11 shows the dose response for killing of T47D-Luc cells by BHPIand by the active (−)-105 enantiomer of C-105. The Trypan Blue celldeath assay was used to compare the ability of the active (−) enantiomerof C-105 and BHPI to kill estrogen receptor α (ERα) positive T47D, humanbreast cancer cells expressing luciferase (T47D-Luc cells). 300,000T47D-Luc cells/well in 10% FBS were plated in a 6 well plate intriplicate. 24 hours after plating, the indicated concentrations of theactive (−) enantiomer of C-105 [(−)-105] or BHPI, or vehicle [Veh] wereadded to the cells. After 24 hours, the cells were harvested, and the %dead cells was determined by automated Trypan Blue exclusion using theCountess II automated hemocytometer. Percentage of viable cells wascalculated by doing 100−(Cell Death %). (n=3±SEM).

FIG. 12 shows the dose response of (−)-105 is superior to BHPI inkilling MCF-7 and TYS-Luc cells. The Trypan Blue cell death assay wasused to evaluate the ability of the active enantiomer of 105 (−)[(−)-105] to kill ERα positive MCF-7 and TYS-Luc, human breast cancercells. 300,000 T47D cells/well in 10% FBS were plated in a 6 well platein triplicate. 24 hours after plating, the indicated concentrations ofBHPI, or the active enantiomer 105 (−) of C-105 [(−)-105] was added.After 24 hours, the cells were harvested, and the % dead cells wasdetermined by automated Trypan Blue exclusion using the Countess IIautomated hemocytometer. Percentage of viable cells was calculated bydoing 100−(Cell Death %). (n=3±SEM).

FIG. 13 shows the dose response study of (−)-105 is superior to BHPI andcurrent endocrine therapies in killing TDG-Luc cells. In the top figure,the dose response shows that (−)-105 is superior to BHPI in killingTDG-Luc cells. The lower left figure shows current endocrine therapies,ICI/Fulvestrant/Faslodex and z-4-hydroxytamoxifen (the active form oftamoxifen) do not kill TDG-Luc Cells. The Lower right figure shows the(+)-105 enantiomer is inactive and does not kill TDG-luc cells. TheTrypan Blue cell death assay was used to evaluate the ability of theactive enantiomer of C-105 [(−)-105] to kill ERα positive TDG-Luc cells.For all studies, 300,000 TDG-Luc cells/well in 10% FBS were plated in a6 well plate in triplicate. 24 hours after plating, the indicatedconcentrations the active enantiomer of (−)-105, or BHPI (top) ICI, orOHT (Lower left) or (+)-105 (lower right) were added. After 24 hours,the cells were harvested, and the % dead cells was determined byautomated Trypan Blue exclusion using the Countess II automatedhemocytometer. Percentage of viable cells was calculated by doing100−(Cell Death %). (n=3±SEM).

FIG. 14 shows the IC₅₀s for killing of cancer cells by (−)-105. Celllines: ERα positive breast cancer: T47D, MCF-7 TYS (T47D Y537S) TDG(T47D D538G), BT-474; ERα negative MDA-MB-231 breast cancer cells; 6,000cells/well were plated (96 well plate) in 100 μl of medium. DMSO Conc.was adjusted to 1% in each well. (−)-105 at concentrations from0-100,000 nM was added. After 24 hours Alamar Blue reagent (10 μl of 0.1mg/ml) was added. After 2-4 hours fluorescence in the wells was read(λ_(ex): 555 nM, λ_(em): 585 nM, Spectra Max M3 Plate reader). %relative death was determined by setting the reading for 10,000 nMraptinal-treated cells to 100% dead cells and the reading forvehicle-treated cells to 0% dead cells. (n=3±SEM).

FIG. 15 shows long-term experiments wherein (−)-105, but not BHPI, kills100% of TYS-Luc cells. To simulate the effect of several weeks oftreatment of a tumor in a mouse xenograft, long-term cell cultureexperiments were performed. TYS-Luc cells (T47DERαY537S-Luc cells;ERαY537S is the most lethal ERα mutation seen in metastatic breastcancer) were plated at 4,000 cells/well in 96 well plates. Cells wereinitially maintained for 2 weeks in medium containing either DMSOvehicle, 1 μM inactive (+) enantiomer of C-105, 1 μM BHPI, or 1 μM ofthe active (−) enantiomer of C-105 (Treat). After 2 weeks (medium changeevery 2 days) cell number was determined using MTS and a standard curveof absorbance versus cell number for TYS-Luc cells. Notes: (i) Barsdenote the BHPI and (−)-105 arms of the experiment. No drug is presentduring regrow. (ii) Because absorbance values are extremely low, the MTSassay cannot accurately quantitate fewer than 200 cells/well (iii) Afterseparation, the inactive enantiomer still contains ˜1-2% activeenantiomer; thus, it retains some activity when used at the high conc.of 1,000 nM), Another set of similarly treated wells was not assayedwith MTS and after 4 washes with PBS was placed in estrogen-free mediumcontaining 10% cd-FBS for 4 weeks (medium change every 2 days) (Regrow).This allows any surviving cells to regrow. After 4 weeks the cells wereassayed using MTS. Notably, there was no regrowth of cells treated withactive (−)C-105 and robust regrowth of BHPI-treated cells. (Note: Visualinspection of the wells confirmed that there were no cells in the welltreated for 2 weeks with active (−)-105.) To test whether the cells thatregrew after BHPI treatment were resistant to BHPI they were re-treatedfor another cycle (BHPI Retreat) and shown to retain sensitivity to BHPIkilling. (n=8 biological replicates±SEM.

FIG. 16 shows long-term experiments that simulate therapy, wherein(−)-105, but not BHPI, eradicates, lethal, therapy resistant breastcancer cells. Cells: MCF-7Y537S (MCF-7 ERαY537S-luciferase (derived fromMCF-7 human breast cancer cells. MCF-7 cells are the most widely line ofhuman breast cancer cells. This is the most lethal and second mostcommon mutation in metastatic breast cancer.) Experimental conditionsare as in the legend to FIG. 15. (+)-105 enantiomer was inactive.

FIG. 18 shows (−)-105 kills breast cancer cells resistant to killing byBHPI. ERα containing T47D human breast cancer cells able to survive in 1μM BHPI were selected by clonal outgrowth. The effect of 100 nM and 1 μMBHPI and (−)-105 on growth of the (BHPI-sensitive) parental T47D cellsand partially BHPI-resistant T47D clones 1,3,8, and 11 was evaluated.4,000 cells/well were plated. Cell number was by MTS assay from astandard curve of absorbance versus cell number. Conclusions: BHPIblocked growth and killed many of the T47D cells and (−)-105 killed allthe T47D cells. BHPI inhibited growth but could not stop growth of theresistant clones; (−)-105 completely blocked the growth of the resistantclones and reduced their cell numbers below the original cell number of4,000 cells/well. Thus, in 4 days (−)-105 killed some, but not all, ofthe resistant cells.

Concerning the mechanism of action of −105 for the anticipatory unfoldedprotein response (UPR), FIG. 19 shows (−)-105 potently induces formationof spliced XBP-1 mRNA. ERα positive T47D-luciferase, human breast cancercells, were maintained for the indicated times in vehicle (Ctl,Control), 100 nm BHPI or 100 nM (−)-105. At the indicated times RNA wasisolated and levels of sp-XBP1 mRNA quantitated by qRT-PCR. (n=3±SEM).(−)₁₀₅ or BHPI activation of the UPR activates the UPR sensor IRE1α,resulting in cleavage of inactive XBP1 mRNA to active spliced (sp)-XBP1mRNA.

Increased sp-XBP1 mRNA is a widely used marker for unfolded proteinresponse (UPR) activation. Thus, (−)-105 is a much stronger UPRhyper-activator and inducer of sp-XBP 1 than BHPI.

FIG. 20 shows (−)-105, but not BHPI induces near-quantitative,inhibition of the synthesis of new proteins. ERα positive Caov-3, humanovarian cancer cells, were treated for the indicated times with 1,000 nmBHPI or 1,000 nM (−)-105. Cells were briefly labeled with³⁵S-methionine. Incorporation of labeled ³⁵S-methionine into protein wasdetermined by trichloroacetic acid precipitation and trapping ofprecipitated protein, but not free amino acids, in small Whatmanhardened ashless filters. After solubilizing the protein in the filterswith base and neutralization with acid, the samples were counted in aliquid scintillation counter. Shown is the average (n≥3) percentinhibition of protein synthesis compared to the 0-time sample. The nearquantitative inhibition of protein synthesis by (−)-105 is consistentwith (−)-105 inducing lethal hyperactivation of the PERK arm of the UPR.Even non-growing cells are constantly degrading protein and making newproteins. Cells in which nearly all (>90%) of protein synthesis isinhibited cannot grow and will ultimately die.

FIG. 21 shows blocking the efflux of calcium from the endoplasmicreticulum with 2-APB inhibits (−)-105-induced cancer cell death. ERαpositive MCF-7 and TYS-Luc were treated with DMSO vehicle (Veh), or BHPIor (−)-105 plus or minus 2-APB for the indicated times. Cells death wasdetermined using the instrument-based Trypan Blue exclusion assay. At 30minutes, 2-APB nearly completely blocked (−)-105-induced cell death andat 45 and 60 min. it partially blocked cell death. This is consistentwith (−)-105 inducing powerful lethal activation of the anticipatory UPRpathway. (n=3±SEM).

FIG. 22 shows −105 reduces intracellular ATP levels; this reduction inATP levels is blocked by inactivating the SERCA pump with thapsigargin.TYS-Luc cells were maintained DMSO vehicle (Veh), 1,000 nM BHPI, 1,000nM −105 with and without 10,000 nM thapsigargin (THG) for the indicatedtimes. ATP levels were determines using a kit and relative ATP levelswere from a standard curve. Note that at 1 hr, THG completely blockedthe −105-induced decline in intracellular ATP levels. (n=3±SEM).

FIG. 23 shows blocking the decline in ATP levels with thapsigargininhibiting −105 induced cell deaths. TYS-Luc cells were maintained inDMSO vehicle (Veh) or BHPI, or (−)-105 with or without thapsigargin(THG) for the indicated times. Blocking the decline in ATP levels (FIG.19) with THG inhibited (−)-105-induced cell death. Note that when THGwas present, there was a strong inhibition of cell (−)-105-induced celldeath at 1 hour. (n=3±SEM).

Example 3. Additional Data for Compounds Related to 105

FIG. 24 shows a comparison of the ability of test compounds and BHPI tokill TDG cells. TDG cells were incubated for 24 hours with 75 nM of BHPIor 75 nM of each of the indicated test compounds: 4, 6 and (±)-105(enantiomers not separated). Cell death was determined using theinstrument-based Trypan Blue exclusion assay. Structures are in theSynthesis section. Conclusion: Compared to BHPI, compounds 4 and 105exhibit a greatly increased ability to kill the TDG cells. (n=3±SEM).

FIG. 25 shows a comparison of the ability of test compounds and BHPI tokill TYS cells. TYS cells were incubated for 24 hours with 35 nM of BHPIor 35 nM of each of the indicated test compounds:2, 4, and 105. Celldeath was determined using the instrument-based Trypan Blue exclusionassay. (n=3±SEM). Thus, compared to BHPI, compounds 4 and 105 exhibit agreatly increased ability to kill the TYS cells.

FIG. 26 shows a comparison of the ability of test compounds 4, 6, 105and BHPI to kill TYS cells. TYS cells were incubated for 24 hours with50 nM of of BHPI or 50 nM of each of the indicated test compounds: 4, 6and 105. Cell death was determined using the instrument-based TrypanBlue exclusion assay. (n=3±SEM). Thus, compared to BHPI, compounds 4 and105 exhibit increased ability to kill the TYS cells.

FIG. 27 shows a comparison of the ability of test compounds and BHPI tokill T47D cells. T47D cells were incubated for 24 hours with 75 nM ofBHPI or 75 nM of each of the indicated test compounds: 4 and 6. Celldeath was determined using the instrument-based Trypan Blue exclusionassay. (n=3±SEM). Thus, compound 4 displays a greatly increased abilityto kill breast cancer cells containing wild type estrogen receptor.

In FIG. 28, neither the test compounds nor BHPI kill non-tumorigenic ERαNegative MCF-10A breast cells. ERα Negative MCF-10A cells weremaintained for 24 hours in DMSO vehicle or 75 nM of BHPI or 75 nM ofeach of the indicated test compounds: 4, 6 and 105. Cell death wasdetermined using the instrument-based Trypan Blue exclusion assay.(n=3±SEM). Thus, at 75 nM, neither BHPI nor any of the test compoundsinduce significant death of the ERα Negative MCF-10A cells.

FIG. 29 shows the evaluation of the ability of test compounds and BHPIto kill T47D cells. T47D cells were treated for 24 hours with 100 nMBHPI, or with 100 nM of each of the test compounds: 6, 8, and 105. (n.s.not significant). Cell death was determined using the instrument-basedTrypan Blue exclusion assay. (n=3±SEM). Thus, compared to BHPI,compounds 105 displays increased ability to kill T47D cells.

FIG. 30 compares the ability of BHPI and test compounds to inhibitproliferation of T47D, TYS and TDG cells. T47D cells were in mediumcontaining 10 FBS (which contains estrogen), TYS-4 and TDG-1 cells werein medium containing 10% cd-FBS (estrogen depleted). Cells are plated at2,000 cells/well in 96 well plates. After 24 hours in medium containingFBS or charcoal-dextran treated FBS (depleted of endogenous estrogens),the indicated concentration of each compound (in DMSO at 1/1000^(th) ofthe volume of medium) was added. After 2 days the medium was changed,and the compounds were added again. After 2 additional days (4 daystotal) MTS assays are used to evaluate cell proliferation. (n=6±S.E.M.).Thus, BHPI and the new compounds effectively inhibit proliferation ofthe T47D, TYS-4 and TDG-1 cell. The data suggests that compounds 2, 4, 6and 105 are more effective than BHPI in killing these ERα positivebreast cancer cells.

Additional data are shown in FIG. 31 and FIGS. 33-43. Through amedicinal chemistry campaign, (±)-1 was discovered to show an unexpectedcytotoxic phenotype (FIG. 36A). All known targeted therapies forestrogen receptor alpha (ERα) are cytostatic, meaning they only preventcell growth and do not kill cancer cells. Preparative chiral columnchromatography provided access to enantiomerically pure material(Example 4) identified by polarimetry as (−)-105 and (+)-105. Chemicalderivatization and x-ray crystallography further characterized theactive enantiomer having the (R)-configuration and the inactiveenantiomer having the (5)-configuration (Scheme 5). Biological data(FIG. 36B and Example 4) demonstrate that (R)-1 is cytotoxic to ERαpositive cells (MCF-7) with minimal effects seen in ERα negative cells(MDA-MB-231). Therefore, the compound was renamed as SERK-F6 forSelective Estrogen Receptor Killer with this version having sixfluorines). The dose dependent activity of SERK-F6 was determinedagainst a panel of cancer cell lines (FIG. 36C and FIG. 37A) for ERα.

An important aspect for cytotoxic therapy is the ability toquantitatively kill a cell population. SERK-F6 quantitatively kills ERαcell lines in a rapid 24-hour crystal violet assay (FIG. 36D) and inlong-term cell culturing experiments (FIG. 36E-F and FIG. 37B). SERK-F6kills via anticipatory unfolded protein response (UPR) activation (FIG.38). Important markers of this mechanism are the increased levels ofspliced XBP1 (sp-XBP1) (FIG. 38A), rapid decreased in cellular ATPlevels (FIG. 38B), and rapid inhibition of protein synthesis (FIG. 38C).

The pharmacokinetics of SERK-F6 was measured in mice (FIG. 40A-B).Additional studies showed that SERK-F6 was blood-brain barrier penetrant(FIG. 40C-D). Also, long-term treatment of SERK-F6 does not ablate ERαtissues as measured by circulating estradiol levels (FIG. 40E). Thesedata demonstrate that SERK-F6 can achieve biologically relevantconcentrations in-vivo and be well-tolerated.

To probe the efficacy of SERK-F6 for killing ERα positive tumors, largeMCF-7 tumors were grown and then intervened with therapy. SERK-F6treatment results in dramatic tumor regressions (FIG. 39A and FIG.40F-G). Because current therapy (i.e. fulvestrant, Fulv.) is cytostatic,dramatic effects against these large and established tumors are notobtained. Additionally, daily SERK-F6 treatment did not lead to anychange in murine weight during the study, demonstrating the tolerance ofSERK-F6 in-vivo (FIG. 4011). Furthermore, to demonstrate in-vivomechanism of action of SERK-F6, MCF-7 tumors were grafted in mice andthen treated with SERK-F6. The mice were then sacrificed prior tocomplete tumor eradication. This data showed activation of the UPR withincreases in P-PERK and P-eiF2alpha levels (FIG. 39B-E and FIG. 40I-J).

Because ERα mutations leading to constitutive activation and therapyresistance represent a clinical challenge, it was demonstrated thatSERK-F6 can also address these resistant tumors. Thus, SERK-F6eradicates Y537S tumors (i.e. TYS cell line) in a dose-dependent mannerand in multiple dosing administrations (i.e., i.p. and oraladministration) and was also tolerated (FIG. 41A-D and FIG. 42B-C). Upontreatment cessation, tumors did not regrow, indicating complete tumoreradications (FIG. 42A). With a lower sub-therapeutic dose of SERK-F6(i.e. 10 mg/kg oral), tumors did regrow but retreatment of these tumorswith higher doses of SERK-F6 demonstrated that these regrown tumors arestill sensitive to SERK-F6 and are not resistant (FIG. 42D). This resultshows the importance of complete cell killing for eradicating cancers.SERK-F6 treatment also leads to destruction of D538G mutant tumors (TDGcell line) (FIG. 41D-E). SERK-F6 treatment was again tolerated (FIG.42E) at higher doses and enough to eradicate tumors (FIG. 42F). Whilelower doses do not completely eradicate tumors, retreatment of thesetumors with higher doses of SERK-F6 regresses the tumors (FIG. 42G-H).

To confirm that tumor regressions are not just seen with T47D backgroundcell lines (TYS and TDG), MCF-7 cells were treated with Y537S or D538Gmutations in their ERα proteins (MYS and MDG cells, respectively) withSERK-F6. Dramatic tumor regressions were observed (FIG. 41F-G, and FIG.43A-D). Again, higher doses are necessary for complete tumor responses,while lower doses lead to non-resistant tumor growth (FIG. 43 B-D).SERK-F6 treatment also eradicates metastatic tumor burden (FIG. 41I).

Example 4. Determination of Absolute Configuration and IC50s ofEnantiomers of Compound 1 (105)

Species IC₅₀ (nM) S.E.M. (±)-1 42.6 1.9 (R)-1 20.3 0.9 (S)-1 >1000 —

Preparative gram-scale chiral separation of (±)-1 yields enantiopure(R)-1 and (S)-1 with opposite optical rotations (Scheme 5). IC₅₀ valuesof (±)-1, (R)-1, and (S)-1 against MCF-7 cells reveals (R)-1 is theactive species, referred to as SERK-F6. Values were obtained after24-hour incubation utilizing Alamar blue fluorescence assay normalizedto live and death controls (vehicle and raptinal treated). (S)-1 IC₅₀is >1 μM. Error shown as S.E.M.

Example 5. Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a compound of a formula described herein, a compoundspecifically disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof (hereinafter referred to as ‘Compound X’):

(i) Tablet 1 mg/tablet ‘Compound X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 300.0

(ii) Tablet 2 mg/tablet ‘Compound X’ 20.0 Microcrystalline cellulose410.0 Starch 50.0 Sodium starch glycolate 15.0 Magnesium stearate 5.0500.0

(iii) Capsule mg/capsule ‘Compound X’ 10.0 Colloidal silicon dioxide 1.5Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0 600.0

(iv) Injection 1 (1 mg/mL) mg/mL ‘Compound X’ (free acid form) 1.0Dibasic sodium phosphate 12.0 Monobasic sodium phosphate 0.7 Sodiumchloride 4.5 1.0 N Sodium hydroxide q.s. solution (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(v) Injection 2 (10 mg/mL) mg/mL ‘Compound X’ (free acid form) 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1 N Sodium hydroxide q.s. solution (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL

(vi) Aerosol mg/can ‘Compound X’ 20 Oleic acid 10Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000Dichlorotetrafluoroethane 5,000

(vii) Topical Gel 1 wt. % ‘Compound X’   5% Carbomer 934 1.25%Triethanolamine q.s. (pH adjustment to 5-7) Methyl paraben  0.2%Purified water q.s. to 100 g

(viii) Topical Gel 2 wt. % ‘Compound X’   5% Methylcellulose   2% Methylparaben  0.2% Propyl paraben 0.02% Purified water q.s. to 100 g

(ix) Topical Ointment wt. % ‘Compound X’   5% Propylene glycol   1%Anhydrous ointment base  40% Polysorbate 80   2% Methyl paraben 0.2%Purified water q.s. to 100 g

(x) Topical Cream 1 wt. % ‘Compound X’  5% White bees wax 10% Liquidparaffin 30% Benzyl alcohol  5% Purified water q.s. to 100 g

(xi) Topical Cream 2 wt. % ‘Compound X’   5% Stearic acid  10% Glycerylmonostearate   3% Polyoxyethylene stearyl ether   3% Sorbitol   5%Isopropyl palmitate   2% Methyl Paraben 0.2% Purified water q.s. to 100g

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Compound X’. Aerosol formulation (vi) may be used inconjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

1. A compound of Formula I:

or a salt or solvate thereof; wherein R¹, R², R³ and R⁴ are eachindependently H, halo, —OR^(A), —SR^(A), —N(R^(A))₂, alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl; A¹, A², A³ and A⁴ are eachindependently H, halo, or alkyl; G¹ is halo, —OR^(B), —SR^(B),—S(═O)₂R^(B), or alkyl; G² is halo, —OR^(C), —SR^(C), —S(═O)₂R^(C), oralkyl; X and Z are each independently O, S, or —NR^(D); and R^(A),R^(B), R^(C) and R^(D) are each independently H or alkyl, wherein, whenpresent, —OR^(B) and —OR^(C) are not both —OH; wherein each alkyl,cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substitutedwith one or more substituents.
 2. The compound of claim 1 wherein thecompound is the (S)-enantiomer.
 3. The compound of claim 1 wherein thecompound is the (R)-enantiomer.
 4. The compound of claim 1 whereinR^(A), R^(B), R^(C) and R^(D) are each independently H or —(C₁-C₆)alkyl,and R¹, R², R³ and R⁴ are each independently H, halo, or —(C₁-C₆)alkyl.5. The compound of claim 1 wherein A¹, A², A³ and A⁴ are eachindependently H or halo, and G¹ is —OR^(B).
 6. The compound of claim 1wherein X is —NR^(D) and Z is O.
 7. The compound of claim 1 wherein R¹is CH₃, CH₂CH₃, CF₃, CHF₂, CH₂CF₃, CF₂CH₃, or CF₂CF₃.
 8. The compound ofclaim 1 wherein G¹ is —OR^(B), and R^(B) is CH₃, CH₂CH₃, CF₃, CHF₂,CH₂CF₃, CF₂CH₃, or CF₂CF₃.
 9. The compound of claim 1 wherein thecompound is a compound of Formula II or Formula III:


10. The compound of claim 1 wherein the compound is a compound ofFormula IV:

wherein G¹ is —OR^(B), and R^(B) and R¹ are each independently alkyl orcycloalkyl, wherein the alkyl and cycloalkyl are optionally substitutedwith one or more halo groups.
 11. The compound of claim 1 wherein one ormore hydrogen atoms is deuterium or tritium, one or more carbon atoms isa carbon isotope, or a combination thereof.
 12. The compound of claim 1wherein the compound is any one of compounds (S)- or (R)-2, 4, 6, 8 or105:


13. The compound of claim 12 wherein the compound is levorotatory. 14.The compound of claim 12 wherein the compound is dextrorotatory.
 15. Thecompound of claim 12 wherein the compound is (R)-105 or (S)-105.
 16. Thecompound of claim 15 wherein the compound is (R)-105.
 17. The compoundof claim 1 wherein the compound has a binding affinity for the alphaestrogen receptor (ERα), and the IC₅₀ of the binding affinity is lessthan about 500 nM.
 18. A composition comprising the compound of claim 1and a second drug.
 19. A pharmaceutical composition comprising thecompound of claim 1 in combination with a pharmaceutically acceptablediluent, carrier, excipient, or buffer.
 20. The pharmaceuticalcomposition of claim 19 wherein the compound is a racemic mixture of(R)-105 and (S)-105:


21. A method of treating a cancer comprising administering to an ERαpositive cancer subject in need thereof a therapeutically effectiveamount of the compound of claim 1, thereby treating the cancer in thesubject.
 22. The method of claim 21 wherein the compound kills orinhibits growth of ERα positive cancer by hyperactivation of theunfolded protein response (UPR) in the endoplasmic reticulum.
 23. Themethod of claim 21 wherein the compound is a racemic mixture of (R)-105and (S)-105:


24. The method of claim 21 wherein the ERα positive cancer is a breastcancer, ovarian cancer, uterine cancer, cervical carcinoma, orendometrial cancer.
 25. The method of claim 21 wherein the compound isadministered orally or by injection. 26-27. (canceled)